Diagnostic applications of double D-loop formation

ABSTRACT

The present invention describes the formation of RecA protein catalyzed double-stranded probe:duplex linear target DNA complexes that are stable to deproteinization. The uses of this stable probe:target complex in diagnostic/DNA detection systems in in vitro and in situ DNA hybridization reactions is discussed. The probe:target complexes are also useful for diagnostic application in RecA protein facilitated DNA amplification reactions.

This application is a continuation-in-part of co-owned, U.S. applicationSer. No. 07/910,791, filed 9 Jul. 1992, now abandoned which is acontinuation-in-part of co-owned, U.S. application Ser. No. 07/755,462,now U.S. Pat. No. 5,273,881 filed 4 Sep. 1991, which is acontinuation-in-part of co-owned, U.S. application Ser. No. 07/520,321,now U.S. Pat. No. 5,223 ,414 filed 7 May 1990.

FIELD OF THE INVENTION

The present invention relates to the formation of RecA-catalyzed stabledouble D-loop structures that can be utilized in a variety of diagnosticmethods including two probe capture/detection systems, RecA-facilitatedDNA amplification, and in situ hybridization.

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BACKGROUND OF THE INVENTION

RecA+ protein (wild type) is a 37,842 dalton protein found in thebacterium Escherichia coli, which is important for homologous DNArecombination. Most information about its biochemistry and enzymologycomes from in vitro studies using purified RecA+ protein. Numerous invitro studies have shown that RecA+ protein is intimately involved inthe pairing reaction between homologous DNA sequences that ultimatelyleads to homologous recombination events (Radding; see Cox et al. orRoca et al. for recent reviews of RecA+ protein properties). It is thispairing reaction that makes RecA+protein highly useful for DNAdiagnostics applications.

In the presence of ATP, RecA+ protein catalyzes strand exchange betweena number of substrates, the most relevant for DNA probe applicationsbeing single- and double-stranded DNAs. RecA protein coatedsingle-stranded DNA probes interact with the homologous portion of adouble-stranded ("native") target sequence initially by forming arecombination intermediate containing hybridized, partially joinedmolecules called (single) D-loops (or in some cases triple-strandedstructures) (Shibata et al., 1979). This is followed by branchmigration, and forming of fully hybrid molecules between the originalsingle- and double-stranded DNAs, depending upon the extent of theirhomology.

Short displacement loops or triple-stranded D-loop structures in lineartargets are usually unstable after deproteinization. RecA protein hasbeen shown to form stable complexes with short oligonucleotides, between9 and 20 bp (or larger) in length, in the presence of ATPγS and excessRecA protein (Leahy et al.). When linear double-stranded targets areused, stable probe target pairing after RecA removal appears to require(i) a homologous region of at least 38 to 56 bp, and (ii) the locationof the probe target homology at the end of the linear duplex (Hsieh etal. 1990; Gonda et al.).

Rigas et al. reported that a single-stranded 43-mer could form a singleD-loop complex stable to deproteinization when double-strandednegatively supercoiled circular plasmid DNA was used as the target.

When a double-stranded negatively supercoiled circular target DNA isused, RecA coated single-stranded oligonucleotide probes can also bestabilized by psoralen crosslinking before removal of the RecA protein:probe-target single D-loop products can be recovered if the oligos areat least 30-mer size (Cheng et al., 1989). To obtain psoralen crosslinkstabilized single D-loop probe-target complexes when double-strandedlinear DNA duplexes are used as target DNA, the probes must be at least80 to 107-mer size (Cheng et al., 1988): these reactions are very lowefficiency when compared to similar reactions with negativelysupercoiled circular targets.

Experiments performed in support of the present invention havedemonstrated that probe:target DNA complexes, which are stable todeproteinization, can be generated in RecA protein catalyzed reactionsproviding that duplex probes, which contain sequences complementarybetween probe-strands, are used in the hybridization reactions. Thisdiscovery provides a number of opportunities for diagnostic applicationthat exploit this stable RecA protein catalyzed probe:targethybridization complex.

SUMMARY OF THE INVENTION

The present invention includes a diagnostic method for detecting alinear duplex DNA analyte, having first and second strands, where theanalyte contains a first internal DNA target sequence. The methodteaches providing a set of two DNA probes that each contain sequencescomplementary to the first target sequence strand or the second targetsequence strand, where these probes also have a region of complementaryoverlap to each other. Both probe strands are then coated with RecAprotein in a RecA protein coating reaction. The RecA coated probes arecombined with the linear duplex DNA, which contains the target sequence,under conditions that produce a probe:target complex. This probe:targetcomplex contains both probe strands and both strands of the linearduplex analyte. The probe:target complex is stable to deproteinization,although in the method of the present invention it is not necessarilydeproteinized. The presence of the probe DNA in the probe:target complexis then detected.

In one embodiment of the invention the RecA protein is the wild-typeRecA protein of Escherichia coli. Alternatively, the RecA protein can bethe mutant recA-803 protein of Escherichia coli or a RecA-like proteinfrom a number of sources.

The RecA-protein coating reactions of the present invention can becarried out using a variety of co-factors, including ATPγS, rATP (aloneand in the presence of a regenerating system), dATP, GTPγS, and mixes ofATPγS and rATP, and ATPγS and ADP.

In one embodiment of the invention, the region of complementary overlapbetween the probe strands is at least about 78 base pairs and less thanabout 500 base pairs. The probe strands may also contain an end terminalextension of DNA that is not complementary to either target strand. Whenboth strands contain such an end terminal extension, these DNAextensions may be complementary to each other.

One way in which to accomplish the detecting of the method of thepresent invention is by deproteinization of the probe:target complex,followed by electrophoretic separation of the probe:target complex fromfree probe. The probe:target complex can be deproteinized by a varietyof methods including treatment with SDS or proteinase K, as well asstandard chemical deproteinization methods, such as phenol-basedmethods. Alternatively, the detecting can include the use of a capturesystem that traps the probe:target complex, where the first probe strandis labeled with a capture moiety and the second probe strand is labeledwith a detection moiety. For example, one probe strand can be biotinlabeled and the other digoxigenin labeled. The probe:target complex canthen be captured/detected using solid support-streptavidin (oravidin)/labeled anti-digoxigenin, or solid support-anti-digoxigeninantibody/labeled streptavidin (or avidin). In a different embodiment,the first probe strand contains a capture moiety and the second probestrand contains a radioactive label to be used for detection.

The probe strands can be labelled for capture in a number of ways, forexample, using biotin or digoxigenin attached to the probe andstreptavidin (or avidin) or an anti-digoxigenin antibody, respectively,for capture. The probe strands can also be labelled for detection usinga number of different moieties including: radioactive, biotin,digoxigenin. Radioactive labels can be identified by, for example,autoradiography or scintillation counting. The presence of biotin ordigoxigenin can be detected by streptavidin or an anti-digoxigeninantibody, respectively, where the streptavidin (or avidin) oranti-digoxigenin is radioactively labeled, enzyme labeled (e.g.,alkaline phosphatase, peroxidase, beta-galactosidase or glucose oxidase)or fluorochrome-labeled (e.g., fluorescein, R-phycoerythrin, orrhodamine). Detection of the probe strands in the probe:target complexcan also be accomplished by DNA polymerase facilitated primer extensionfrom the 3'-ends of each probe strand, where the primer extension isperformed in the presence of all four dNTPs and one or more dNTPcontains a detectable moiety.

The method of the present invention further includes providing a secondset of two DNA probes, having first and second strands, complementary toa second duplex target sequence, where the first strand of the probecontains sequences complementary to one strand of the second targetsequence and the second strand of the probe contains sequencescomplementary to the other strand of the second target sequence, where(i) these probes also have a region of complementary overlap to eachother, and (ii) the second set of probes does not hybridize to the firstset of probes. The two probe sets are coated with RecA protein in a RecAprotein coating reaction. The RecA coated probe sets are combined withthe linear duplex DNA, containing the two target sequences. Thecombining is done under conditions that produce probe:target complexeswhich contain all four probe strands. The resulting probe:target complexis stable to deproteinization. The presence of the probe DNA is thendetected in the probe:target complexes.

The method involving two probe sets can be utilized in many of the sameways as described above for a single probe set. For example, the firstprobe set can be labeled with a capture moiety and the second probe setlabeled with a detection moiety.

The double-stranded probe:duplex target complexes involving two probesets can also be used in a RecA protein facilitated DNA amplificationmethod. For example, the two probe sets can be hybridized to theirduplex target sequences in the presence of ATPγS or rATP (with orwithout a suitable ATP regeneration system), dATP, and mixtures of ATPγSand ADP! and reacted in a reaction mixture also containing, all fourdNTPs, RecA protein and DNA polymerase. This reaction is performed belowthe temperature required for thermal dissociation of the two targetstrands and continued until a desired degree of amplification of thetarget sequence is achieved. The amplification reaction may furtherinclude repeated additions of (i) DNA polymerase and (ii) RecAprotein-coated probes during the course of the amplification reaction.Other approaches to amplification, which can be applied to the presentinvention, have been set forth in co-pending U.S. application Ser. No07/520,321, filed 7 May 1990. In each probe set, the 3' end of onestrand will be internal to the region defined by the two primer sets:these ends are necessary for the amplification reaction. However, theopposite 3'ends of each primer pair, external to the region defined bythe two primer sets, can be blocked to inhibit the formation ofextension products from these ends. This amplification method can alsobe used as a detection method, where detection of the probe in theprobe:target complex is accomplished by DNA polymerase facilitatedprimer extension from the 3'-ends of each probe strand, where the primerextension reaction is performed in the presence of all four dNTPs andone or more dNTP contains a detectable moiety.

The double-stranded probe:duplex target complexes can also be used toblock cleavage of any targeted restriction site. Blocking cleavage canbe accomplished in a number of ways including: (i) forming theprobe:target complex and treating with the restriction enzyme beforedeproteinization of the complex; (ii) using methylated or un-methylatedprobes, depending on the sensitivity of a selected enzyme to thepresence of methyl groups; and (iii) introducing a sequence mismatch ineach strand of the probe, which, when the probe hybridizes to thetarget, eliminates the restriction site.

The double-stranded probe:duplex target complexes can also be used togenerate site specific cleavage of double-stranded target DNA. Thedouble-stranded probe can be modified with moieties capable of cleavingeach strand of the target duplex: this probe modification can take placebefore or after deproteinization depending of the nature of the cleavingmoiety. Examples of such moieties are iron FeII (for iron/EDTAfacilitated cleavage), non-specific phosphodiesterases, and restrictionendonucleases. In either case, cleavage specificity is conferred by thetarget sequence which is defined by the double-stranded oligonucleotideprobe.

Both the restriction site protection method and the site specificcleavage method are useful in restriction fragment length polymorphismanalysis.

Another embodiment of the present invention includes a method forisolating a linear duplex DNA analyte, having first and second strands,containing a first internal DNA target sequence, where the duplex DNAanalyte is present in a mixture of nucleic acid molecules. In thismethod a set of two DNA probes is provided, having first and secondprobe strands, where the first and second probe strands (i) containcomplementary sequences to the first and second target sequence strands,and (ii) where these complementary sequences also contain complementaryoverlap between the probe strands. The probes are then coated with RecAprotein. The coated probes are combined with the linear duplex DNA,which contains the target sequence, under conditions that produce aprobe:target complex containing the probe strands and both targetstrands: the resulting probe:target complex is stable todeproteinization. The probe:target complex is separated from the mixtureof nucleic acid molecules. The duplex DNA analyte, which contains thetarget sequence, is then isolated.

In this method, the complex can be separated from the nucleic acidmixture using, for example, probes containing biotin moieties that arecaptured with streptavidin or avidin. The streptavidin or avidin can bebound to a solid support, such as paramagnetic beads.

The method further includes heat denaturation of the isolatedprobe:target complex at a temperature (i) sufficient to release theduplex DNA analyte containing the target sequence from the complex, and(ii) below the melting temperature of the duplex DNA analyte containingthe target sequence. This allows the isolation of intact duplexmolecules. The duplex can then be denatured to single-strands if sodesired. Alternatively, the complex can be heat denatured at atemperature (i) sufficient to release the duplex DNA analyte containingthe target sequence from the complex, and (ii) at or above the meltingtemperature of the duplex DNA analyte containing the target sequence.This results in the isolation of single-stranded DNA molecules derivedfrom the captured duplex.

Another embodiment of the present invention is a method for detecting alinear duplex DNA analyte in a mixture of nucleic acid molecules. Themethod includes isolating the linear duplex DNA analyze as describedabove and obtaining single-stranded DNA molecules derived from duplexDNA analyte: this is typically accomplished by heating the duplex abovethe melting temperature of the duplex (heat denaturation). To thesingle-stranded target DNA analyte molecules, at least one DNA synthesisprimer is added that is complementary to the target sequence and thatdoes not contain sequences which were present in either of the twooriginal DNA probes. Detection of the DNA analyte is accomplished by DNApolymerase facilitated primer extension from the 3'-end of the primer,wherein the primer extension is performed in the presence of all fourdNTPs and at least one dNTP contains a detectable moiety.

The double-stranded probe:duplex target complexes of the presentinvention can also be used for diagnostic in situ detection techniques.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the relationships of the probes and primers, listedin Table 1, to the lambda genome.

FIG. 2 presents the nucleotide sequence of a 500 bp lambda genomicregion: this sequence is also presented as SEQ ID NO:1.

FIG. 3 shows an autoradiogram of a DNA band-shift gel electrophoresisassay to illustrate RecA protein binding to DNA probes.

FIG. 4A shows an ethidium bromide stained gel on which the components ofdeproteinized hybridization reactions using 500-mer and 280-mer probeswere resolved. FIG. 4B shows an autoradiograph of the gel shown in FIG.4A.

FIG. 5A shows an ethidium bromide stained gel in which the components ofdeproteinized hybridization reactions using 280-mer, 121-mer, and 79-merprobes were resolved. FIG. 5B shows an autoradiograph of the gel shownin FIG. 5A.

FIG. 6A shows an ethidium bromide stained gel in which the components ofdeproteinized hybridization reactions using differentially labeled121-mer DNA probes were resolved. FIG. 6B shows an autoradiograph of thegel shown in FIG. 6A. FIG. 6B illustrates that two DNA probe strands arenecessary for the production of stable, deproteinized, RecA proteincatalyzed hybridization complexes.

FIG. 7 illustrates a model of stable double-stranded probe:duplex lineartarget DNA complexes.

FIG. 8 shows a gel from which stable double-stranded probe:duplex lineartarget DNA complexes were isolated, where the duplex probe strands weredifferentially labeled.

FIG. 9 illustrates a variety of double-stranded probe:duplex lineartarget DNA complexes.

FIGS. 10A, 10B, and 10C illustrate several detection systems based on asingle double-stranded probe:duplex linear target DNA complex.

FIGS. 11A and 11B illustrate several detection systems based on amultiple double-stranded probe:duplex linear target DNA complex.

FIG. 12 shows RecA-protein catalyzed two-double D-loop primerpositioning on native target DNA (FIG. 12A) and DNA amplification withligation wise followed by ligation with DNA ligase (in the absence ofprimer/probe displacement) (FIG. 12B).

FIG. 13 shows a DNA polymerase mediated signal amplification reactionusing a single double D-loop probe (FIG. 13A) or multiple double D-loopprobes (FIG. 13B). In FIG. 13, X and X' can be the same or different:for example X can be radioactively labeled and X' can carry adigoxigenin moiety.

FIG. 14 illustrates a detection system involving the use of restrictionendonuclease cleavage of non-target complexed double-stranded probewhere capture of the resulting product is accomplished before (FIG. 14B)or after (FIG. 14A) restriction enzyme digestion.

FIG. 15 illustrates the protection of a restriction site by eithermethylation or RecA protein. In the case of methylation protection thedouble D-loop complex is deproteinized before restriction endonucleasedigestion (FIG. 15A). In the case of RecA protein protection the doubleD-loop complex is deproteinized after restriction endonuclease digestion(FIG. 15B).

FIG. 16 shows an ethidium bromide stained agarose gel on which thecomponents of deproteinized RecA mediated double D-loop hybridizationreactions, using heat denatured 280-mer probe and different cofactors,were resolved by electrophoresis.

FIG. 17 shows an autoradiogram of the gel shown in FIG. 16, afterdrying.

FIG. 18 shows an ethidium bromide strained agarose gel on which thecomponents of deproteinized RecA mediated double D-loop hybridizationreactions, using heat denatured 500-mer probe and ATPγS and ATPγS/rATPmixes as cofactors, were resolved by electrophoresis.

FIG. 19 shows an autoradiogram of the gel shown in FIG. 18, afterdrying.

FIGS. 20A, 20B, and 20C show a schematic outline of a homogeneousdiagnostic assay for duplex DNA target with Double-D-loop hybrids.

DETAILED DESCRIPTION OF THE INVENTION

I. Generation of RecA Catalyzed Probe:Target Hybridization ComplexesWhich are Stable to Deproteinization.

A. DNA probes and primers.

Experiments performed in support of the present invention show thatshort double-stranded DNA molecules or complementary single-strandedmolecules can be used to generate hybridization complexes with lineartarget DNA molecules at internal regions and these complexes are stableto deproteinization. As an example of these stable hybridizationcomplexes, double-stranded and complementary single-stranded DNAmolecules of varying lengths were prepared for use as probes and primers(Example 1, Table 1). These DNA molecules were chosen to have homologyto various portions of a 500 bp region of the lambda phage genome. Therelationships of the probes and primers, listed in Table 1, to thelambda genome is illustrated in FIG. 1. The nucleotide sequence of the500 bp lambda genomic region is presented in FIG. 2.

B. Preparation of RecA protein and Probe Coating.

In the present invention RecA protein refers to a family of RecA-likerecombination proteins all having essentially all or most of the samefunctions, particularly: (i) the protein's ability to properly positionprimers on their homologous targets for subsequent extension by DNApolymerases; (ii) the ability of RecA protein to topologically prepareDNA for DNA synthesis; and, (iii) the ability of RecA protein/DNA primercomplexes to efficiently find and bind to complementary sequences. Thebest characterized RecA protein is from E. coli; in addition to thewild-type protein a number of mutant RecA-like proteins have beenidentified (e.g. recA-803, Madiraju, et al.). Further, many organismshave RecA-like strand-transfer proteins (e.g., Fugisawa, H., et al.;Hsieh, P., et al., 1986; Hsieh, P., et al., 1989; Fishel, R. A., et al.;Cassuto, E., et al.; Ganea, D., et al.; Moore, S. P., et al.; Keene, K.,et al.; Kimeic, E. B., 1984; Kimeic, E. B., 1986; Kolodner, R., et al.;Sugino, A., et al.; Halbrook, J., et al.; Eisen, A., et al.; McCarthy,J., et al., Lowenhaupt, K., et al.)

RecA protein is typically obtained from bacterial strains thatoverproduce the protein: Example 2 describes the purification ofwild-type E. coli RecA protein and mutant recA-803 protein from suchstrains. Alternatively, RecA protein can also be purchased from, forexample, Pharmacia (Piscataway N.J.).

The conditions used to coat DNA probes with RecA protein and ATPγS aredescribed in Example 3. Alternatively, probes can be coated using GTPγS,rATP (alone or in the presence of a rATP regenerating system (BoerhingerMannheim)), dATP, mixes of ATPγS and rATP, or mixes of ATPγS and ADP.The use of ATPγS, rATP, dATP and GTPγS as cofactors in the RecA proteincoating reaction is described in Example 10. The results of doubleD-loop complex formation using these cofactors are presented in FIGS. 16and 17. FIG. 17 shows that in the presence of each of ATPγS, rATP, dATPand GTPγS double D-loop hybridization complexes, which are stable todeproteinization, were formed. Further, Example 11 describes the use ofmixtures of ATPγS/rATP as cofactors for the RecA protein coatingreactions. The results shown in FIGS. 18 and 19 show that in thepresence of such mixtures double D-loop hybridization complexes, whichare stable to deproteinization, were formed. In addition to ATPγS/rATP,mixtures of other cofactors also work in the RecA protein coatingreaction.

The coating of probes with RecA protein can be evaluated in a number ofways. First, protein binding to DNA can be examined using band-shift gelassays (McEntee et al.). Example 3 describes the use of the DNAband-shift gel assay to illustrate RecA protein binding to DNA probes.Labelled probes were coated with RecA protein in the presence of ATPγSand the products of the coating reactions were separated by agarose gelelectrophoresis: FIG. 3 shows an autoradiogram of the resulting DNA inthe gel. The data presented in FIG. 3 illustrates that followingincubation of RecA protein with denatured duplex probe DNAs the RecAprotein effectively coats single-stranded DNA probes derived fromdenaturing the duplex probe. As the ratio of RecA protein monomers tonucleotides in the probe increases from 0, 1:27, 1:2.7 to 3.7:1 for121-mer (lanes 1-4, respectively) and 0, 1:22, 1:2.2 to 4.5:1 for159-mer (lanes 5-8, respectively), DNA probe's electrophoretic mobilitydecreases, i.e., is retarded, due to RecA-binding to the DNA probe. Thepartial retardation of the DNA's probe mobility observed in lanes 2,3and 6,7 reflects the non-saturation of probe DNA with RecA protein.Thus, as expected (Leahy et al.), an excess of RecA monomers to DNAnucleotides is required for efficient RecA coating of short DNA probes.

A second method for evaluating protein binding to DNA is the use ofnitrocellulose filter binding assays (Leahy et al.; Woodbury et al.).The nitrocellulose filter binding method is particularly useful indetermining the dissociation-rates for protein:DNA complexes usinglabeled DNA. In the filter binding assay, DNA:protein complexes areretained on a filter while free DNA passes through the filter. Thisassay method is more quantitative for dissociation-rate determinationsbecause the separation of DNA:protein complexes from free probe is veryrapid.

Typically, to perform such filter binding assays nitrocellulose disks(Schleicher and Schuell, BA85 filters or HAW POO25 nitrocellulosefilters) are pretreated, soaked in buffer and then placed on a vacuumfilter apparatus DNA:protein binding reactions are often diluted toreduce the concentration of components without dissociating complexes.The reactions are passed through the discs with vacuum applied. Underlow salt conditions the DNA:protein complex sticks to the filter whilefree DNA passes through. The discs are placed in scintillation countingfluid (New England Nuclear, National Diagnostics, Inc.), and the cpmdetermined using a scintillation counter.

C. DNA Targets.

To study the specificity of the RecA catalyzed hybridization of probeswith homologous double-stranded linear DNA targets at internal sites,several model lambda DNA target systems were used including thefollowing:

(1) A mixture of 14 DNA fragments, ranging in size from 92 to 8598 bpgenerated by DraI (Promega) restriction enzyme digestion of completelambda genomic DNA (48.5 kb; Bethesda Research Laboratories,Gaithersburg Md.). The target fragment homologous to the region definedin FIG. 1 and 2 is a 8370 bp DraI fragment. The region(s) of homology tothe probes, listed in Table 1, were all at least 832 bases in from the3' end of the double-stranded target DNA fragment.

(2) A mixture of two fragments (38,412 and 10,090 bp) generated by ApaIrestriction enzyme digestion of complete lambda genomic DNA. Theapproximately 10 kb fragment contains the region defined in FIGS. 1 and2. This region of homology lies at least 2460 bp from the 3' end of thedouble-stranded target DNA fragment.

(3) The 10 kb fragment of an ApaI lambda DNA digest, agarose gelpurified and deproteinized.

(4) A double digest of lambda with DraI and BamHI in which the targetDNA fragment is 2957 bp, with probe target homology at least 832 basesfrom the 3' end of the double-stranded target DNA fragment.

(5) Whole lambda viral DNA was also used as target. In this case,probe:target homology was at least 7131 bases from the 5' end of thewhole lambda genome.

D. RecA-Facilitated Formation of Hybridization Complexes BetweenDouble-stranded Probe and Target DNA Sequences.

The mixing of RecA coated single-stranded DNA probes and target DNAinitiates the search for homology between RecA coated DNA probes andduplex target DNA molecules. In the case of a single probe sequence,once the RecA:DNA probe filament is formed it can catalyze the searchfor homology and D-loop formation between complementary probe and targetDNA sequences. Traditional single D-loops can be formed betweensingle-stranded RecA-coated DNA probes with about 500 bases or less ofhomology with linear double-stranded target DNAs. These D-loops areunstable after protein removal when the position of probe:targethomology is at an internal position on the linear target.

Experiments performed in support of the present invention havedemonstrated that 500-mer and smaller probes can form stabledeproteinized RecA catalyzed double D-loop probe:target complexes atinternal sites on duplex linear DNA targets. However, to form suchstable structures, at least two probes must be used that haveoverlapping complementary sequences to each other. The two probes areRecA coated single-stranded DNA probes and are used in RecA catalyzedprobe:target hybridization reactions,

Example 4 describes the formation of RecA protein-mediated doubleD-loops, or multiplexes. The 500- and 280-mer probes were RecAprotein-coated and the target DNA was the 8369 bp lambda DNA DraIfragment described above. The RecA protein to probe-nucleotide ratio was1.5:1 for 500-mer and 1.3:1 for 280-mer. The double-stranded DNA probeto homologous double-stranded DNA target fragment ratio was 11:1 for500-mer and 22:1 for 280-mer. FIG. 4A shows an ethidium bromide stainedDNA gel on which the DNA components of the deproteinized hybridizationreactions were resolved.

FIG. 4B shows an autoradiograph of the DNA in the gel shown in FIG. 4A.The results presented in FIG. 4B show formation of 500-mer:target and280-mer:target DNA hybridization products that are stable todeproteinization. A comparison between the reactions with and withoutRecA protein, shows that RecA is required for the formation ofhomologous probe:target DNA complexes.

The pUC18 double-stranded circular DNA was included as a positivecontrol in Example 4. Negatively supercoiled double-stranded DNAcircular molecules are known to form probe:target RecA protein catalyzedhybridization products with short single-stranded probes that are stableto deproteinization (Rigas et al.; and Cheng et al., 1988).

In order to confirm the identity of the hybridization products formed inthe above experiment, RecA protein coated probes were reacted withtarget fragments derived from DraI/BamHI double digest of lambda genomicDNA. In this experiment, the homologous target fragment generated by thedouble digest was 2957 bp in length and the position of probe:targetsequence homology was unchanged from the previous experiments (i.e., 832bs from the 3' end of the homologous target fragment). The hybridizationreactions were performed under identical conditions to those justdescribed for the 8370 bp lambda DNA DraI target fragment.

Electrophoretic separation followed by autoradiographic analysis ofthese RecA protein catalyzed hybridization reactions showed thatdeproteinized probe:target DNA complexes now migrated to the position ofthe 2957 bp target fragment, confirming that the probe:target DNAhybridization reaction was indeed with specific homologous targets.

The RecA protein catalyzed probe:target reactions were carried out inthe presence of an excess of non-homologous linear DNA target molecules.

Example 5 describes the formation of complexes stable todeproteinization between small double-stranded probes and lineardouble-stranded target DNAs. In the hybridization reactions presented inExample 5, denatured probes were coated at a RecA protein toprobe-nucleotide ratio of 1.8:1, 0, 5.7:1, 5.9:1, 2.6:1 and 11.8:1,lanes 1-6, respectively in FIGS. 5A and 5B. The double-stranded probe todouble-stranded target fragment ratios were 4.8:1 (280-mer), 3.6:1(121-mer), and 5.2:1 (79-mer). FIG. 5A shows DNA from an ethidiumbromide stained gel on which the components of the deproteinizedhybridization reactions were resolved.

FIG. 5B shows an autoradiograph of the gel shown in FIG. 5A. The resultspresented in FIG. 5B show that the stable deproteinized hybridizationprobe:target product can be formed with probes shorter than 280 bases insize. The addition of too much RecA protein appears to decrease theamount of stable product formed in the DNA hybridization reaction(compare lanes 4, 5 and 6). Because the 121-mer and 79-mer probes usedin this experiment were derived from restriction enzyme digestion of ³²P end-labeled 280-mer and 500-mer duplex probes, each DNA probecontained either the 5' strand or the 3' strand labeled, not both, aswith the 280-mer: the 5' and 3' ends of molecules are identified withrespect to whole lambda DNA. The signals observed in lanes 3-6 of FIG.5B show that either the 5' or the 3' probe strand can take part in theprobe:target reaction: this observation is consistent with theconclusion that both probe strands are involved in the formation of theprobe:target DNA hybridization complex that is stable todeproteinization.

Numerous hybridization experiments following the basic protocoldescribed in Example 4 and 5 have confirmed that the RecA proteincatalyzed hybridization reaction can occur under a broad range ofreaction conditions. Typically, when different concentrations of targetDNA are used, the yield of deproteinized hybrid is proportional to theamount of homologous target DNA in the reaction. Some reactionsconditions can be summarized as follows:

(i) ATPγS concentrations between 1 and 12 mM were tested in probeRecA-coating reactions. This concentration range of ATPγS gave stablehybridization products after target addition: the preferred range wasabout 2.4 to 8 mM. ATPγS, rATP (alone and in the presence of aregenerating system), dATP, GTPγS, and mixes of ATPγS and rATP, alsowork in probe coating reactions (Examples 10 and 11). Further, whencommercial preparations of ATPγS are used in a reaction, the purity ofthe preparation can vary from preparation to preparation. ATPγS obtainedfrom Pharmacia are usually approximately 95-97% ATPγS. ATPγS obtainedfrom Sigma vary between approximately 75% to approximately 90% ATPγS:these preparations usually contain between approximately 10% to 20% ADP.ATPγS from both Pharmacia and Sigma sources have been tested:preparations from both of these sources work well in RecA double D-loopreactions. Thus, combinations of ATPγS and ADP also work in RecAmediated double-D-loop hybridization reactions. Further, DNA probes wereeffectively coated with RecA protein in the presence of a mixture ofATPγS and rATP, preferred mixtures contained about 1.4 and 1 mM of eachcomponent, respectively. The results of these experiments show that RecAcan use a wide variety of cofactors and cofactor combinations for doubleD-loop complex formation.

(ii) Mg⁺⁺ acetate concentrations in the final reaction containing theprobe and target DNAs worked overt a broad range of Mg⁺⁺ concentrations:4 to 25 mM, with the preferred range being about 6 to 8 mM;

(iii) RecA protein concentrations between 8.4 to 41 μM were tested inprobe coating reactions: each concentration was active;

(iv) RecA protein to probe-nucleotide ratios during probe coatingbetween 1:3 and 6:1 were effective with the preferred range beingbetween about 2:1 and 4:1 ratios;

(v) Final (micromolar) double-stranded DNA probe to double-stranded DNAtarget molecule ratios were between 2:1 and 22:1 all yielded stabledeproteinized probe:target hybrids;

(vi) The DNA hybridization reaction works in an analogous Tris-HClreaction buffer (pH 7.5), although probe coating and strand transfer inacetate buffer appears to give more products than the Tris system;

(vii) recA-803 mutant protein was active in forming stable hybridizationcomplexes;

(viii) The hybridization reaction functions in the presence ofsingle-strand binding (SSB) protein (Morrical et al.);

(ix) RecA protein coated single-stranded DNA probes, including mixturesof coated-denatured-double-stranded probes, stored at -20° C. forseveral days were active in hybridization complex formation afterincubation with target at 37° C.;

(x) The hybridization reaction can be carried out with whole lambdagenomic DNA as target. The probe:target hybridization reactions can bealso be carried out when the target DNA is embedded in agarose plugs ormicrobeads: for example, stable double D-loop hybrids have been formedusing RecA-protein coated probes with intact 48.5 kb λ DNA targetsembedded within agarose plugs;

(xi) The region of complementary overlap between the probe strandstypically is about 79 base pairs and less than about 500 base pairs.Probes with this degree of complementary overlap form stable products atinternal target sites in the RecA-catalyzed hybridization reaction ofthe present invention. Generation of stable hybridization products wasalso demonstrated at the ends of linear molecules (for example, using80-mer probes and the 500-mer duplex as target (FIG. 1)). Standardprobe-strand to target-strand complementarity is between 90-100%.However, RecA protein is known to catalyze the formation ofhybridization complexes containing some non-specific base pairinteractions (Cheng et al. 1989). Accordingly, probe:targetcomplementarity can be reduced depending on probe size and the requiredspecificity of the detection reaction: typically complementarity is notlower than 70% base pair matches between each probe-strand andtarget-strand.

(xii) Probes having less than about 79 base pairs of overlap can be usedin the present invention: stabilization of the double D-loop, subsequentto deproteinization, may be advantageous when probes of these smallersizes are used. One method of further stabilizing the double D-loopcomplexes is psoralen cross-linking (Cheng et al., 1988): suchcross-linking is particularly useful in situ since it permits the use ofharsh washing conditions.

The results presented in FIG. 5B indicated that the observedprobe:target products were stable to deproteinization because both DNAprobe strands were present on the same target molecule. Onerepresentation of such a stable complex is shown in FIG. 7. Thisstructure is referred to herein as a double D-loop or multiplex DNAstructure as opposed to the traditional single D-loop, ortriple-stranded displacement loop or triplex structure (two targetstrands and a single DNA probe complementary to a particular singletarget strand).

Example 6 presents data that confirms that two RecA protein-coated DNAprobe strands are required for the production of stable deproteinizedprobe:target hybridization products on a linear target DNA molecule atan internal region of DNA homology. Individual 121-mer probe strandswere chemically synthesized to insure that individual probe strandswould not be contaminated with small amounts of the complementary(opposite) DNA strand. In order to distinguish the presence of each ofthe two individual complementary DNA probe strands, the probes weredifferentially labeled: one strand with a 5' terminal ³² P label and theother with a single 5' terminal biotin label.

Since only one strand was radioactively labeled the ³² P specificactivities of each double D-loop DNA hybridization reaction were thesame: accordingly, comparison between the results of all experiments wasmore convenient. Hybridization reactions were performed as described inExample 6. FIG. 6A shows an ethidium bromide stained gel on which theDNA components of the deproteinized hybridization reactions wereresolved.

FIG. 6B shows an autoradiograph of the DNA in the gel shown in FIG. 6A.The results in FIG. 6B show that two probe strands are required forstable deproteinized probe:target hybrid production. In addition, thereaction works whether both probes are coated with RecA protein togetheror in separate reactions. Further, the hybridization reaction generatesdeproteinized stable complexes even when the DNA probes are added to thereaction sequentially (lanes 5 and 6). The addition of the ³² P strandfirst to the reaction mix appears to provide more DNA hybridizationproduct. It is possible that the terminal biotin label is slightlyinhibitory due to the size of the chemical spacer arm or the position ofthe label on the probe. However, regardless of the order of probeaddition to the hybridization reactions, two probe strands are requiredto generate stable deproteinized homologous complexes. The RecA-mediatedhomologous probe targeting reaction can also use probes containingbiotin incorporated at internal positions. Such probes can besynthesized using a modification of polymerase chain reaction (Mullis;Mullis, et al.) where bio-14-dATP replaces a certain percentage (e.g., 5to 25%) of the dATP normally used during synthesis.

The rate of RecA-facilitated homologous pairing of short DNA probes totheir cognate target sequences has been shown to be positively relatedto the length of attached heterologous DNA tails (Gonda et al.).Accordingly, probes used in the hybridization reactions of the presentinvention may include heterologous tails, i.e., terminal sequences thatare non-homologous to the target DNA, in order to speed the homologouspairing of the probe sequence to the target sequence.

E. Capture/Detection System.

The presence of both the ³² P- and biotin-labeled 121-mer probe strandson the same target molecule was further confirmed using acapture/detection system (Example 7). The deproteinized double D-loopproducts were captured using streptavidin-magnetic beads. Capture of thebiotin containing probe simultaneously captured the ³² P-labelled probe.FIG. 8 shows the DNA from the gel from which the probe:target complexeswere isolated before streptavidin capture of the biotin moiety. The DNAcomplexes were isolated by extraction from gel fragments correspondingto the expected size of the probe:target complex (Example 7). Theextracted DNA was then exposed to streptavidin-coated paramagneticbeads. The beads were then isolated and placed in scintillation fluidfor detection of the ³² P-labelled DNA probe strand. The results of thisanalysis are presented in Table 2. The data show that only reactionsusing two probe strands and RecA protein give a capture signal abovebackground. This experiment used isolated DNA target migrating at the 10kb target DNA position for capture, thus ruling out the possiblepresence of complex recombination products between multiple 10 kbtargets that could be captured and detected without actually having adouble D-loop structure on an individual 10 kb target molecule. Further,the hybrid molecules formed in these reactions are quite stable underthe isolation conditions used supporting the conclusion that thecaptured ³² P signal was not an artifact of complementary probereassociation.

II. Utility

FIG. 9 shows a number of possible double D-loop structures. FIG. 9Arepresents the formation of a double D-loop structure at an internalsite on a DNA target molecule. FIG. 9B represents a similar structureexcept that the probe DNA molecules have been tailed with heterologousDNA (Gonda et al.). Such tailing can serve several purposes: (i)facilitating RecA loading onto small probes; (ii) providing an extensionmolecule for the inclusion of labels in the probe, for example,digoxigenin or biotin; (iii) providing a capture sequence; and (iv)providing a sequence to hybridize to an additional reporter molecule.

FIG. 9C represents the situation where two probes are used that have aregion of complementary overlap (i.e., a region in which they arecomplementary to each other) in addition to homologous terminalextensions (i.e., regions complementary to the target DNA but not to theother probe).

FIG. 9D represents the situation where two probes are used that have aregion of complementary overlap in addition to heterologous terminalextensions (i.e., regions not complementary to the target DNA but not tothe other probe). FIG. 9F shows a similar situation where heterologousterminal extensions are present at both the 5' and 3' ends of each probestrand. FIG. 9G illustrates the situation where the homologous tails arecomplementary to each other, but not to the target DNA.

The double D-loop structures need not be composed of only two probes.For example, FIG. 9E shows a double D-loop structure generated from 5separate probe strands: the internal probe strands have regions ofcomplementary overlap to more than one other probe strand. The totalregion of complementary overlap is typically 79 to 500 base pairs, but,as discussed above, this region may be smaller.

The structures in FIG. 9 illustrate several, but not all, possiblecombinations of probe and target DNA that can generate double D-loopstructures stable to deproteinization. One common feature ofdouble-stranded probes to be used in double D-loop reactions is a regionof complementary overlap between the probe strands.

The ability to form stable RecA protein catalyzed deproteinized doubleD-loop probe:target complexes at internal sites allows specificidentification of homologous linear DNA targets. This double D-loopreaction provides new possibilities for hybridization diagnostics. Theassay provides the advantages that differentially labeled complementaryprobe strands can be used in a single reaction and only one small targetsequence needs to be known.

Reassociation of complementary probes is inhibited when saturatinglevels of RecA protein are used (Bryant et al.). Reassociation of suchprobes is also reduced by the inclusion of ATPγS as a cofactor in theprobe coating reactions.

As described above, the complexes of the present invention, which areformed between the RecA protein coated probes and target DNAs, arestable to deproteinization reactions (as described). In someapplications, however, the removal of RecA protein from the complexes isnot required for practicing the application. In such cases the onlylimitation is that the remaining protein molecules do not interfere withthe application (e.g., see Section F below).

A. Target DNAs.

The method of the present invention can be used to diagnose infectiousdisease caused by organisms in clinical samples. These organisms can bediagnosed by the detection of specific DNA characteristic of thecausative organism. Such organisms include the following: bacteria, likeSalmonella, Neisseria, Chlamydia, Shigella, and Streptomyces; viruses,like Herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), andadenovirus, all double-stranded DNA viruses; parasites, like Plasmodiumand Giardia; and mycoplasma, like Mycoplasma pneumonia, M. genitaliumand Pneumocystis.

For any diagnostic assay, probe sequences are chosen to a known regionof homology in the target DNA. Target DNA can be prepared from a numberof different sources by standard techniques: for example, aqueoussolutions, mammalian tissue, cultured cells, plant tissue, bacteria,yeast, blood and blood components (Ausubel et al.; Maniatis et al.;Sambrook et al.; Davis et al.; Fey; Strickler et al.; Kingston;Wachsmuth).

In general, the detection methods of the present invention can beapplied to the detection of duplex DNA in any nucleic acid sample.Applications other than clinical diagnosis of infectious diseasesinclude (i) screening cultured mammalian cells for the presence ofcontaminants, such as mycoplasma (Zlvin et al.), (ii) diagnosis ofcertain genetic diseases caused by specific deletions/mutations,insertions or rearrangements in mammalian DNA, such as α-thalassemia,β-thalassemia, or chronic myelocytic leukemia and (iii) hybridizationprobes to distinguish the presence or absence of a given target sequencein a duplex DNA molecule.

B. The Use of One Double D-loop Structure for Diagnostic Applications.

FIG. 10 illustrates several embodiments of the use of one double D-loopstructure for the isolation and identification of correspondingsequences in duplex DNA targets. One probe can be labeled with a capturemoiety (e.g., as was done with biotin in Example 7). The other probe isthen labeled with a detection moiety, such as a radioactive label,biotin, or digoxigenin or other modified bases. The probes are coatedand hybridized to the nucleic acid sample that is being tested for thepresence of the target sequence. The hybridization reactions can bedeproteinized or used directly.

The probe labeled with the capture moiety is trapped. This trapping canbe accomplished by, for example, labeling the probe with a biotin moietyand exposing the reaction mixture to streptavidin that has been attachedto a solid support. Alternatively, the capture moiety can be digoxigeninand the trapping can be accomplished using an antidigoxigenin antibodyattached to a solid support. Additional groups can be convenientlyattached to the ends of DNA molecules as follows. The oligonucleotideprobe is combined with digoxigenin-11-dUTP (an analog of dTTP,2'-deoxy-uridine-5'-triphosphate, coupled to digoxigenin via an 11-atomspacer arm, Boehringer Mannheim, Indianapolis IN) and terminaldeoxynucleotidyl transferase (GIBCO BRL, Gaithersburg, Md). The numberof dig-11-dUTP moieties incorporated using this method appeared to beless than 5 (probably only 1 or 2). Alternatively, dig-11-dUTP moietiescan be incorporated into the oligonucleotide sequence of a probe as withbiotin.

Typically, the following combinations of double-stranded probes are usedin the capture detection system: (i) first probe/capture, labeled withbiotin or digoxigenin, second probe/detection, radiolabeled; (ii) firstprobe/capture, labeled with biotin, second probe/detection, labeled withdigoxigenin; or (iii) first probe/capture, labeled with digoxigenin,second probe/detection, labeled with biotin.

One convenient method to sequester captured DNA is the use ofstreptavidin-conjugated superparamagnetic polystyrene beads as describedin Example 7. After capture of DNA, the beads can be retrieved byplacing the reaction tubes in a magnetic rack.

Alternatively, avidin-coated agarose beads can be used. Biotinylatedagarose beads (immobilized D-biotin, Pierce) are bound to avidin.Avidin, like streptavidin, has four binding sites for biotin. One ofthese binding sites is used to bind the avidin to the biotin that iscoupled to the agarose beads via a 16 atom spacer arm: the other biotinbinding sites remain available. The beads are mixed with hybridizationcomplexes to capture biotinylated DNA (Example 7). Alternative methods(Harlow et al.) to the bead capture methods just described include thefollowing streptavidinylated or avidinylated supports: low-proteinbinding filters, or 96-well plates, or modified biotin capture methodssuch as iminobiotin (Rigas, B., et al.).

For either of the above bead methods, the beads are isolated and theamount of hybridization complex that has been captured is quantitated.The method of quantitation depends on how the second strand DNA probehas been prepared. If the second probe is radioactively labelled thebeads can be counted in a scintillation counter. Alternatively, thecaptured DNA may be detected using a chemiluminescent, fluorescent orcolorimetric detection system.

Many of the experiments described above have made use of radio-labelledoligonucleotides: Example 7 combines the use of a biotin labeled firstprobe with a radioactively labelled second probe. The techniquesinvolved in radiolabeling of oligonucleotides have been discussed above.A specific activity of 10⁸ cpm per μg DNA is routinely achieved usingstandard methods (e.g., end-labeling the oligonucleotide with adenosineγ-³² P!-5' triphosphate and T4 polynucleotide kinase). This level ofspecific activity allows small amounts of DNA to be measured either byautoradiography of gels or filters exposed to film or by direct countingof sample in scintillation fluid.

Radiolabeling and chemiluminescence (i) are very sensitive, allowing thedetection of sub-femtomole quantities of oligonucleotide, and (ii) usewell-established techniques. In the case of chemiluminescent detection,protocols have been devised to accommodate the requirements of amass-screening assay. Non-isotopic DNA detection techniques haveprincipally incorporated alkaline phosphatase as the detectable labelgiven the ability of the enzyme to give a high turnover of substrate toproduct and the availability of substrates that yield chemiluminescentor colored products.

For chemiluminescent detection, biotinylated or digoxigenin-labelledoligonucleotide probes can be detected using the chemiluminescentdetection system "SOUTHERN LIGHTS", developed by Tropix, Inc. The basictechnique can be applicable to detect DNA that has been captured oneither beads, filters, or in solution.

Alkaline phosphatase is coupled to the captured DNA complex. To do thisseveral methods, derived from commonly used ELISA (Harlow et al.;Pierce, Rockford Ill.) techniques, can be employed. For example, thesecond strand DNA probe can be end-labelled with digoxigenin-11-dUTP(dig-11-dUTP) and terminal transferase (as described above). After theDNA is captured and removed from the hybridization mixture, ananti-digoxigenin-alkaline phosphatase conjugated antibody is thenreacted (Boehringer Mannheim, Indianapolis Ind.) with thedigoxigenin-containing oligonucleotide. The antigenic digoxigenin moietyis recognized by the antibody-enzyme conjugate.

Captured DNA hybridization products are detected using the alkalinephosphatase-conjugated antibodies to digoxigenin as follows. Onechemiluminescent substrate for alkaline phosphatase is3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD). Dephosphorylation of AMPPD resultsin an unstable compound, which decomposes, releasing a prolonged, steadyemission of light at 477 nm. Light measurement is very sensitive and candetect minute quantities of DNA (e.g., 10² -10³ attomoles).

Colorimetric substrates for the alkaline phosphatase system have alsobeen tested. While the colorimetric substrates are useable, use of thelight emission system is more sensitive.

An alternative to the above biotin capture system is to use digoxigeninin place of biotin to modify the first strand probe: biotin is then usedto replace the digoxigenin moieties in the above described detectionsystem. In this arrangement the anti-digoxigenin antibody is used tocapture the DNA hybridization complex. Streptavidin conjugated toalkaline phosphatase is then used to detect the presence of capturedoligonucleotides.

Other alternative capture systems include the following: (i) the use ofa DNA binding protein and its cognate binding sequence, where thecognate binding sequence is the capture moiety that is included as a 5'terminal sequence in the first strand probe-(Kemp et al.); and (ii) theuse of hybridization capture where a non-target-complementary DNAsequence, such as poly(T), is incorporated as a 5' terminal sequence inthe first strand probe, and a complementary nucleic acid, such aspoly(A), is used to capture the probe and associated nucleic acid byhybridization. Either of these two methods can be used in conjunctionwith a solid support.

Another alternative system is to fix one probe to a membrane (Saiki etal.), coat the probe, add target and the second coated probe,deproteinize, wash and detect.

FIG. 10 illustrates several arrangements for one double D-loop structuredetection. FIG. 10A shows one probe strand labeled with a capturemoiety, such as biotin, and the second probe strand labeled with adetection moiety, such as digoxigenin. FIG. 10B shows one probe strandlabeled with a capture moiety, such as digoxigenin, and the second probestrand having a homologous tail extension that is labeled with multipledetection moieties, such as biotin. FIG. 10C illustrates the addition ofdetection (or capture moieties) to heterologous tails attached to eitherand/or both strands of the double-stranded probe.

C. The Use of Multiple Double D-loop Structures for DiagnosticApplications.

In addition to exploiting one double D-loop structure complexes incapture/detection systems, multiple double D-loop structures can be usedas well. In cases where reassociation of probe strands may be a problem,for example with larger probes, the use of multiple double D-loopstructures provides for a reduced background. In this method two or moresequences need to be known in the target sequence.

FIG. 11 illustrates several arrangements for detection based on twodouble D-loop structures. FIG. 11A shows an example of labeling bothstrands of a first duplex probe with the capture moiety and both strandsof a second duplex probe with a reporting/detection group. The capturemoiety can be contained in either one or both strands of the first probeset. In this system the hybridization complexes are captured asdescribed above, however, reassociation of the strands of the firstduplex probe generates no background for the reaction since neitherstrand contains a reporter/detection group. After capture, thehybridization complexes are detected on the basis of the presence of thesecond duplex probe in the hybridization complex. Detection of thereporter group is accomplished as described above.

A second embodiment of this system, based on the presence of multipledouble D-loop structures in the target complex, is illustrated in FIG.11B. In this case only one strand of the first duplex probe is labeledwith the capture moiety and only one strand of the second duplex probeis labeled with the reporter moiety.

As described above for one double D-loop structure detection,heterologous tails and sequences homologous to the target DNA can beadded to the duplex probes.

D. The Use of Double D-loop Structures in RecA protein Facilitated DNAAmplification Reactions.

DNA amplification reactions have been described that predominantly relyon thermal denaturation (Mullis; Mullis et al.; Scharf et al.) forstrand separation in preparation for continued amplification. Describedbelow are two detection systems based on DNA amplification subsequent tothe formation of single or multiple double D-loops.

(i) One amplification/detection method of the present invention utilizesmultiple double D-loop structures to facilitate amplification withoutthe need for thermal denaturation.

Experiments performed in support of the present invention havedemonstrated that use of two pairs of complementary DNA primers, whichare homologous to different regions of the double-stranded target, inthe hybridization reactions of the present invention results in theformation of two double D-loops in the target DNA. These double D-loopsflank and define a specific region of DNA on a native duplex target(FIG. 12A): DL801/2 and DL803/4 (Table 1) are examples of probes/primersthat can participate in this reaction.

The resulting DNA target structure is recognizable by various DNApolymerases as a substrate for DNA synthesis. An example of one suchamplification reaction is presented in Example 8. The substrate for theamplification reaction described in Example 8 is the lambda DNA genome.The primers define a target region of approximately 500 bp. Typically,the amplificationreactions contain DNA polymerase (e.g., the Klenowfragment), RecA protein coated primers, ATPγS (or rATP (alone and in thepresence of a regenerating system), dATP, GTPγS, and mixes of ATPγS andrATP or ATPγS and ADP), the target substrate, necessary cofactors andall four dNTPs (including modified or labeled dNTPs). These reactionsmay also contain DNA helicase, topoisomerase, or other similar DNAunwinding agents and/or other DNA polymerases.

These reaction conditions favor extension of the primers at their 3'ends with subsequent primer elongation in the 5' to 3' direction. The 3'end extension of two or more of the four DNA primers in the structure bypolymerase defines regions of DNA target amplification between DNAprimers (see FIG. 13). DNA polymerase extension of the other two primerscan also occur at their 3' ends, unless these are chemically orphysically blocked to restrict DNA amplification to a defined region(Example 8).

DNA polymerases catalyzing 3' extension between primer pairs maydisplace primers on the same strand, or, alternatively, new synthesizedproduct(s) could be ligated to the primer with DNA ligase, includingthermoresistant or thermosensitive DNA ligases (Epicentre Technologies,Madison, Wis.). Replication can also be facilitated by addingappropriate DNA helicase, topoisomerase, single-strand binding proteins(SSB), gene 32 (or other similar) proteins, or RecBCD-encoded enzymes orother proteins, some of which have associated helicase activities. Anyprimers properly positioned by RecA protein could be used forreplication initiation.

Typically, probes used in the two-double D-loop RecA protein catalyzedDNA amplification are approximately 60 to 80 bp in size. Probes largeror smaller can also be used as well, but reaction conditions may need tobe modified, for example, inclusion of stabilizing peptides (e.g., SSBor gene 32 protein), drugs, antibodies, polyamines or cross-linkingreagents.

Multiple primers of different sizes can also be used. Primers can alsobe homologous to the target DNA duplex along their entire length, orthey can contain end or internal regions of partial non-homology, suchas heterologous tails (see above). The only requirement for theseprimers is that the bases used for the 3' extension of the desiredamplification product is available to prime DNA synthesis. As describedabove, primers can also contain modified phosphate backbones or basessuch as biotin or digoxigenin or appended functions such as DNAmodifying enzymes and chemical agents.

Reaction in the presence of excess RecA protein-coated primers allowsformation of new multiple or double D-loops on the newly replicated andamplified DNA. The RecA protein-coated primers serve to initiateadditional rounds of DNA synthesis, and in this way the DNA target isamplified without the need for thermally denaturing the target DNA toposition the amplification primers.

DNA amplification reactions can use any of a number of DNA polymerasesor polymerase mixtures, including the following: Klenow large fragmentof E. coli DNA polymerase I; T7; T4; and/or other viral or cellular DNApolymerases and their mutants, for example, double-Klenow mutantproteins having no exonuclease activity.

The DNA products of two-double D-loop reactions are defined by the DNAprimers used. The left and right primers to the double D-loop regions tobe amplified define the 5' ends of the newly synthesized DNAamplification products. When the primers are not displaced, the newlysynthesized DNA product can be ligated in a RecA-catalyzed amplificationreaction as illustrated in FIG. 12B.

Using multiple primer sets, it is also possible to generate DNAamplification products which have cohesive ends. The DNA products canthen hybridize through their overlapping cohesive ends and the length ofthese associated DNAs is subsequently extended (Haase et al.).Elongation of existing strands, displacement synthesis, andRecA-catalyzed base pairing in the overlap regions may increase theyield of large DNA targets. This can be important for RecA-catalyzed DNAamplification in cells in situ, which may, under certain conditions(Haase et al.), require large DNA products or appended groups forretention in situ.

The double D-loop reaction, or multiple double D-loops, can be stablyformed in agarose for in situ amplification reactions. Typically, theagarose is of the low-melting temperature variety, although mixtures ofdifferent types of agarose are possible, and the concentration ofagarose is about 0.4-1%. Under these conditions the agarose gel providesa restrictive medium that also allows retention of shorter DNA products:this is particularly useful in in situ reactions (see below).

The RecA-facilitated DNA amplification reaction can be carried out at37° C. as well as at elevated temperatures that are below the thermaldenaturation temperatures of target DNA duplex or primer:target hybrids,for example 50°-60°C. Use of elevated temperatures in these reactionsexpands the repertoire of enzymes available for primer extension and mayallow longer tracts of DNA to be synthesized. The temperature of theamplification reaction will dictate the choice of reaction components:for example, wild-type E. coli RecA-protein coated probes are added tothe target DNA at 37°-39° C., DNA synthesis is then accomplished at50°-55° C. with Thermus aquaticus DNA polymerase, the temperature of thereaction is then lowered to 37°-39° C. and RecA-protein coated probesare added re-added. Alternatively, at high temperaturestemperature-resistant RecA-like proteins could replace the wild type E.coli RecA protein (as discussed in co-pending, co-owned, U.S.application Ser. No. 07/520,321).

The two-double D-loop, or multiple double D-loop, reaction using RecAprotein-catalyzed primer positioning for DNA amplification reactions hasimportant diagnostic applications for DNA detection and amplification insolution diagnostics or in situ diagnostics.

(ii) A second detection method exploiting the double D-loop and DNAamplification uses polymerase addition of labeled or modified dNTPs forsignal amplification. Extension of the 3' end of a primer in a single(triple-stranded) D-loop structure using DNA polymerase was demonstratedby Cheng et al. (1988). After formation of a single double D-loop in atarget DNA molecule DNA polymerase, necessary cofactors, and all fourdNTPs are added to the reaction. Strand extension takes place from the3' ends of either one or both probe strands of the double-stranded probe(FIG. 13A). Alternatively, the 3' end of the strand containing thecapture moiety can be blocked to prevent primer extension. One or moreof the dNTPs is labeled for detection by a standard method (e.g.,biotin, digoxigenin, or, fluorescent or radioactive moieties). Theincorporation of the labeled dNTP results in the amplification of thedetection signal.

This method can be further exploited by using multiple double D-loopstructures to target the region of interest (as described above). The3'-ends of the double-stranded primers external to the target region caneither be blocked (as illustrated in FIG. 13B by an asterisk) or not.This method of signal amplification can be further enhanced by usingmultiple rounds of RecA-facilitated amplification, as described above,in the presence of a labeled moiety.

Target detection with signal amplification can also be accomplished asfollows. A double-D-loop is formed at the target sequence using twoprobe strands, where at least one of the strands contains a capturemoiety. The resulting double-D-loop complex can be deproteinized andcaptured via contacting the capture moiety with a capture medium. Thecaptured complex is released by heating: the complex can be releasedeither as dsDNA or ssDNA. if necessary, e.g., if the complex is releasedas dsDNA, the complex is denatured and the target DNA released. To thismixture DNA synthesis primer(s), which are complementary to a targetsequence, are added. These primers do not contain sequences that werepresent in the original double-stranded probe. DNA polymerase and dNTPs,are then added, under the appropriate buffer conditions, to synthesizeDNA from the hybridized primer(s). This primer-directed templatesynthesis can be carried out in the presence of labeled dNTPs, forexample, radiolabeled dNTPs, biotin-labeled dNTPs, FITC-labeled dNTPs,or other suitably labeled DNA precursors. The inclusion of label allowstarget detection via the appropriate detection systems, such as, afluorometer in the case of FITC-label ed dNTPs.

E. The Use of Double D-loom Structures in RecA Protein Facilitated insitu Hybridization.

Another detection method which utilizes the double D-loop structure isin situ hybridization with fixed cells (Example 9): RecA-facilitated insitu hybridization methods have been described in co-owned PCTInternational Publication NO. WO 93/05177 "In situ HybridizationMethod," published on Mar. 18, 1993. The in situ hybridization of RecAprotein coated duplex probes provides the ability to localize a targetsequence in an isolated, fixed biological structure or within a nucleusor nuclear volume relative to other targeted sequences and/or thenuclear membrane, using a confocal laser scanning microscope (van Dekkenet al.).

One application of the in situ method is described in Example 9D. Inthis method, dividing HEp-2 nuclei are fixed and probed with theReCA/chromosome-X alpha satellite DNA probe complex, and labeled withFITC-avidin. The pattern of probe bining in the dividing nucleus isevaluated using standard light fluorescent or laser scanning microscopictechniques. To localize the bound probe, the same field is viewed byphase contrast microscopy, without changing the focus of the lens. Byoverlaying the resulting two photomicrographs, the relative position ofthe nuclear membrane and nuclear division plane can be seen with respectto the probe-labeled chromosomes.

This aspect of the present invention provides simplified in situprocedures for localizing target sequence(s) in a biological structure.Typically, fixed cells or subcellular structures are probed insuspension or on slides followed by flow-cytometric or microscopicanalysis. The method reduces artifaces by eliminating the need for aheat denaturation step, and allows more rapid and specific detection oftarget sequences. The method can be applied equally well to unique, lowand/or high-copy number target sequences.

In particular, the method allows detection of low-copy sequences withoutthe requirement to first amplify the sequences. Since most gene mappingand chromosomal studies are expected to involve specific, unique, orlow-copy sequences, the present in situ method provides an importantadvantage for gene mapping studies, as well as for diagnosticapplications involving unique or low-copy numbers of normal, mutant orpathogen sequences. Also, the present method allows for determination ofchromosome content by flow cytometric analysis.

One general diagnostic application of this in situ method is for use inmapping a selected gene or regulatory sequence in a chromosome, and/orin a particular region of the chromosome. The target gene may be onewhich (a) generates a selected gene product, (b) is suspected ofperforming a critical cell-control function, such as a cell or viraloncogene, (c) is related to a repeat sequence, (d) is suspected ofcontaining a genetic defect which prevents expression of an active geneproduct, (e) may be related in chromosome position to a marker proberegion with a known map position, and/or (f) may represent an integratedviral sequence.

The probe strands for in situ hybridization can be labeled in a numberof ways including direct labeling with fluorescent moieties likefluorescein-11-dUTP (Boehringer-Mannheim). Further, individual probestrands can be used to generate a coupled-fluorescence system where, forexample, the emission energy of one fluorescent moiety, incorporated inone strand) emits light at the excitation energy of the secondfluorescent moiety, incorporated in the second probe strand. Such acoupled-fluorescence system takes advantage of the proximity of theprobe strands in the double D-loop complex.

When the DNA probes are directed against specific cellular pathogens,typically for detecting the presence of a viral or bacterial pathogen inan infected cell, the fixed labeled cells may be examined by light orfluorescence microscopy to detect and localize infecting pathogens incells. Alternatively, cell infection, and percent cells infected, can bedetermined by fluorescence activated cell sorting (FACS) after in situhybridization of RecA protein coated duplex probes to nuclei or cells insuspension (Trask et al.).

F. The Use of Double D-loop Structures in Restriction Enzyme CleavageBased Detection Systems.

The double D-loop structures of the present invention can be used todetect the presence of target DNA in a sample by introducing alterationsat the target/double D-loop complex which modify, in a detectablemanner, the response of this complex to restriction enzyme digestion.Several examples of such detection systems are described below.

One method of detection that exploits the double D-loop and restrictionenzyme digestion is as follows. A region in the target DNA is chosen asthe double-stranded probe sequence. The probe sequence is modified tocontain an internal restriction site that is not present in the targetDNA. Such a restriction site can be chosen so as to minimize base pairmismatching between the target and the probe (FIG. 14). The doubleD-loop is then formed and the complexes deproteinized. The complexes arethen captured on the solid support and digested with the restrictionenzyme for which the site has been introduced in the probe sequence (inFIG. 14A, PvuIII). Alternatively, the complexes can be digested with therestriction endonuclease before capture (FIG. 14B). Since the PvuIIrestriction site is not reconstituted when the probe is hybridized tothe target sequence, the restriction enzyme will only cleave renaturedprobe:probe complexes, not probe:target complexes. The solid support iswashed and examined for the presence of the detection moiety. Thismethod allows the reduction of any background signal that may begenerated by probe renaturation.

A second detection method works on a similar principle. In this methodthe target DNA is un-methylated and the double-stranded probe DNA ismethylated before RecA protein-coating. The double D-loop complex isformed, the complex captured and deproteinized. The sample is thendigested with, for example, DpnI which cleaves its recognition site (SEQID NO:2) only when the A residue on both strands is methylated. Sincethe methylated restriction site is not formed when the probe hybridizesto the target sequence, DpnI cleavage only occurs when the probes arerenatured. The solid support is washed and examined for the presence ofthe detection moiety. As above, this method also allows the reduction ofany background signal generated by probe renaturation.

The methylation state of the DNA can also be exploited using thetarget/double D-loop complexes as follows. In this method either thetarget DNA is methlated or the double-stranded probe is methylated priorto RecA protein-coating. The double D-loop complex is formed, thecomplex captured and deproteinized. The captured complexes can beisolated from the solid support and split into multiple samples. Onesample is digested with a methylase-sensitive or methylase-dependentrestriction enzyme and another is digested with a methylase-insensitiverestriction enzyme: for example, MboI does not cleave DNA when the Aresidue is methylated and Sau3A I cleaves the same restriction siteindependent of the A residue's methylation state.

These samples can then be size fractionated (e.g., on an agaroseacrylamide gel, or by HPLC) and the banding pattern of the samplescompared. This method allows isolation and subsequent examination ofrestriction fragment length polymorphisms of a chosen fragment between anumber of samples from different sources.

Methylation may also be used to protect a specific restriction enzymesite from digestion (Nelson et al.). If, for example, it was desirableto isolate a MboI fragment spanning a particular region, but internal tothe region an MboI site existed, the fragment could be isolated asfollows. A double D-loop structure is formed at the internal restrictionsite in the target DNA using methylated probes. The target/double D-loopcomplex is deproteinized, digested with MboI and captured (FIG. 15A).This method can be used to (i) examine restriction enzyme polymorphismsat restriction sites adjacent to the protected site in fragmentsobtained from different sources, or (ii) capture and clone a desiredsequence using a restriction enzyme even when an internal cleavage siteis present for that enzyme. The fact that deproteinized double D-loopstructures are susceptible to restriction enzyme cleavage has beendemonstrated by PleI restriction endonuclease site-specific cleavage ofprobe:target complexes formed with 500-mer probes and 2.9-kb homologoustarget fragments.

The double-stranded RecA protein-coated probes can themselves be used toprotect a specific restriction enzyme site from digestion. In this case,the complex is not deproteinized. The target/double D-loop complex isdigested with the restriction endonuclease and captured (FIG. 15B).Alternatively, the double-stranded RecA protein-coated probes can beused to protect a modification site, for example, a methylation site,from modification. In this case, as for the above restriction siteprotection, the complex is not deproteinized. The target/double D-loopcomplex is treated with the modification reagent and then deproteinized.Modification target sites within the target/double D-loop complex areprotected and lack the modification.

The ability to block restriction site cleavage is useful in genomicmapping. For example, if the target DNA defines a known region in thegenome, like the PvuI site at approximately 26.3 kb on the lambdagenome, the known site is protected by one of the approaches describedabove. Unprotected and protected lambda genomic DNA is digested withPvuI. The change in restriction fragment patterns between the twodigests allows one to deduce which fragments are next to oneanother--based on the disappearance of bands and the sizes of the newbands in the sample containing blocked restriction sites.

Restriction enzymes, and their response to methylation states, arecommonly available and conditions for their use are well known in theart (Ausubel et al.; Maniatis et al.).

G. The Use of Double D-loops to Generate Site-Specific Cleavage in DNA.

Oligonucleotides have been used to direct cutting agents to specificsingle-stranded and double-stranded nucleic acids sites (Corey et al.;Dreyer, G. B. et al; Moser, et al). One advantage of oligonucleotidedirected cleavage is that the experimenter is no longer dependent onexisting cleavage functions: any desired DNA cleavage function can betailor-made. The RecA protein-coated double-stranded probes of thepresent invention can be used to generate site specific cleavage in anumber of ways. For example, a specific target sequence is selected anda double-stranded DNA probe corresponding to the selected sequence isgenerated. An EDTA moiety is attached to one or both strands of theoligonucleotide probe. One method of attachment of EDTA to anoligodeoxynucleotide via the C-5 of thymidine has been described byDreyer et al. The probe can then be RecA protein-coated and the doubleD-loop complex formed with target DNA. Cleavage occurs in the presenceof oxygen upon addition of Fe(II) and a reducing agent (usually DTT) tothe EDTA-probe:target hybrids.

Alternatively, cutting can be accomplished using peptide fragmentsderived from DNA binding/cleaving proteins (Sluka, J. P., et al.) whichare attached to the oligonucleotide probes. Further, restrictionendonucleases that have frequent cut sites or relatively non-specificphosphodiesterases, such as staphylococcal nuclease, can be attached toan oligonucleotide to generate a hybrid catalytic agent that hasincreased sequence specificity (Corey et al.).

Oligonucleotides are attached to the phosphodiesterase or other cleavingagent either before RecA protein-coating or after double D-loop complexformation and deproteinization. After association of thephosphodiesterase, nuclease or peptide with the double D-loop complex,the reaction conditions are modified to allow the hydrolysis of bothtarget DNA strands in a site-specific fashion. Depending on the activityof the catalytic agent either one or both strands of the double-strandedprobe is modified to contain the agent.

H. Use of the Double D-loop in DNA Enrichment.

The double D-loop hybridization complex of the present invention canalso be used for separation and enrichment of selected target DNAsequences. For example, the double-stranded probe can be formedcontaining a capture moiety in one or both of the probe strands. Thedouble D-loop complex is formed between the double-stranded probe andtarget DNA contained in a mixture of DNA. The double D-loop complexesthat contain the probe and target sequence are then separated from thereaction mixture using the capture moiety, by, for example, attachmentto a solid support. The complex can then be dissociated by heating torelease the target duplex from the support and, if necessary, thereleased DNA renatured to regenerate the original target duplex DNA.Alternatively, the entire double D-loop complex may simply be releasedfrom the solid support.

To test whether the targeted duplex DNA could be released from thehybrids simply by heating, the thermal stability of deproteinized doubleD-loop hybrids formed with ³² P-end-labeled 500-mer probes and the 10.1kb ApaI target fragment was examined: the hybrids were completely stableat 75° C. in 1×TBE buffer. About half the DNA probe strands werereleased at 80° C. Essentially all of the DNA probe strands werereleased from the target at 85° C. This melting profile is similar tothat for duplex probe:target hybrids formed by heating and then slowlycooling the reactant mixture in the absence of RecA protein. The hybridmelting profile was approximately 10° C. lower than that of duplex500-mer probe, self-annealed, under identical ionic conditions.

The hybridization reaction is potentially useful for targetingchromosomal or gene fragments identified only by sequence-tagged sites(STSs) (Olson, et al.) for the following reasons:

(i) the RecA-mediated hybridization reaction does not requiredenaturation of the duplex DNA target for hybrid formation, and

(ii) targets are released from isolated hybrids by heating to atemperature that dissociates the probe from the probe:target complex butthat does not denature the duplex target-containing analyte, e.g., achromosomal or gene-containing fragment. The target DNA analyte can thenbe recovered in duplex form.

Further, careful control of the hybrid melting temperature would permita selection against the hybrids which might have only partial homologywith the probe. Stringent melting temperature selection may be importantwhen probes are used with complex mixtures of target DNAs. One advantageof recovery of duplex target DNA versus single-strand-denatured targetDNA is that duplex DNA tends to be more resistant to shear forces thantotally denatured single-stranded DNA. The recovery of duplex targetDNA, by the method of the present invention, would allow the enrichmentand isolation of specific duplex gene or genome segments, includinglarge chromosomal fragments, which can then be used for furthermanipulations and/or analysis.

Target DNA duplexes obtained by this method can be used in DNAamplification reactions (Mullis; Mullis et al.) or in standard cloningtechniques (Ausubel et al.; Maniatis et al.).

I. Homogeneous Diagnostic Assay

A protocol for a homogeneous diagnostic assay that can detect a specificnative target DNA duplex has been tested. This assay involvesdouble-stranded target capture using double D-loop hybrids followed byDNA signal amplification, as described briefly below.

(i) Capture of double-stranded DNA targets

A technique can be worked out for using double D-loop hybrids tospecifically capture a large double-stranded DNA such as lambda DNAgenome (-50 kb). The reaction is also applicable for the capture ofsmaller duplex DNA targets. The technique uses RecA-coatedsingle-stranded probes labeled with a capture moiety such as biotin,preferably averaging 300-500 bases in size. All DNA probes are preferredto be homologous to sequences within preferably about 1000 base regionof the duplex DNA target. The biotinylated probes are prepared bynick-translating preferably about 1000 bp DNA duplex fragment in thepresence of bio-14-dATP. Heat-denatured probe DNA is coated with RecAprotein and then reacted with duplex target DNA. After probe:targethybrid formation, the hybridization reaction mixture can be stopped, forexample, with 20 mM EDTA and treated with 0.5M salt, and hybrids can becaptured on washed magnetic Dynabeads® M-280 Streptavidin (Dynal). Aftercapture, beads are washed 3× in buffer containing 1M salt. It is likelythat the high salt conditions at least partially removed a proportion ofthe bound RecA protein. Target DNA capture can be measured by using ³²P-labeled lambda DNA and by directly counting the radioactivity thatremains associated with the beads after washing. The results of thecapture reaction using a large lambda DNA genome (-50 kb) are shown inTable 3. The specificity of the reaction for the capture of biotinylatedprobe which was hybridized with double-stranded target was verified byincluding three control reactions (Table 3, reactions 2-4). Thesereactions showed that: (1) RecA-coated biotinylated probe was requiredfor specific target DNA capture (reaction 1) since no significant signalwas obtained in a reaction without RecA (reaction 2), (2) RecA inclusionin the reaction was not the cause of target capture because onlybackground level DNA capture occurred in the presence ofnon-biotinylated non-homologous DNA probe coated with RecA (reaction 3),and (3) the average background, non-specific, target DNA capture wasapproximately 3.5% (reactions 2-4).

(ii) Signal amplification from captured DNA

A prototype protocol for detecting the bead-captured target DNA isdescribed briefly below. For this step, captured target is released fromthe beads by heating in a reaction mix containing dNTP precursors, oneor more of which is labeled (e.g. bio-14-dATP, or dNTP with a directlydetectable label, such as FITC-11-dUTP, etc . . .) and a ss DNA primer(or primers) not homologous to the original capture probe sequences, buthomologous only to target sequences, is (are) added and allowed toanneal to the single strands of target DNA. After annealing, thereaction is cooled and a DNA polymerase enzyme preferably, DNApolymerase T7 (SEQUENASE™ Version 2.0, USB) and appropriate buffer areadded to allow DNA synthesis by primer extension. Alternatively, a hightemperature polymerase could also be used and the reaction incubated ata temperature allowing processive DNA synthesis. During primerextension, labeled DNA precursor(s) is incorporated into the newlysynthesized DNA strand(s). Because the DNA primer(s) is not homologousto the previously used RecA-coated probe(s), label incorporationspecifically indicates the presence of the correct captured target. Useof a capture moiety either directly associated with the primer(s), suchas poly(A), or able to be added later, allows subsequent capture of theamplified target signal on oligo(dT) attached to magnetic beads,cellulose, etc. Capture is followed by removal of unincorporatedprecursor label from the amplified DNA, and if necessary a blockingagent, such as, for example, I-Block (SOUTHERN-LIGHT™, Tropix) is usedto block the non-specific binding of the detection moiety (e.g.,AVIDx-AP™, Tropix) to the capture matrix. After blocking, substrate isadded to allow specific signal detection from the amplified target DNA,and signal is detected by any appropriate means. A schematic outline ofa homogeneous diagnostic double D-loop assay is shown as follows.Although a specific labeling, capture and detection scheme is presented,there are numerous ways in which such an assay could be practiced.

Schematic outline of a homogeneous diagnostic assay for duplex DNAtargets with Double-D-loop hybrids is shown in FIGS. 20A and 20B.

The following examples illustrate, but in no way are intended to limitthe present invention.

Materials and Methods

Restriction enzymes were obtained from Boehringer Mannheim (IndianapolisInd.) or New England Biolabs (Beverly Mass.) and were used as per themanufacturer's directions.

Generally, oligonucleotides were radiolabeled with γ-³² P!ATP and T4polynucleotide kinase. Labelling reactions were performed in the buffersand by the methods recommended by the manufacturers (New EnglandBiolabs, Beverly Mass.; Bethesda Research Laboratories, GaithersburgMd.; or Boehringer/Mannheim, Indianapolis Ind.). Oligonucleotides wereseparated from buffer and unincorporated triphosphates using Nensorb 20pre-formed columns (NEN-DuPont, Boston, Mass.) as per manufacturer'sinstructions, and subsequently dialyzed versus dd H₂ O if necessary.

EXAMPLE 1 Preparation of RecA-coated Probes

A series of double- and single-stranded DNA probes and primers have beengenerated. The positions of these probes and primers relative to acontiguous 500 base pair region of the lambda phage genome are shown inFIG. 1: the nucleotide sequence of this region of the lambda genome ispresented in FIG. 2. The base positions of the 5' and 3' ends of eachprobe and primer, relative to the lambda viral genome, are listed inTable 1.

                  TABLE 1                                                         ______________________________________                                        LAMBDA BASES DEFINING PROBE AND                                               PRIMER DNA SEQUENCES                                                          Probe or      Bases Included                                                                           Size                                                 Primer        Sequence*  (bs) or (bp)                                         ______________________________________                                        PCR01*        7131-7155  25                                                   PCR02**       7606-7630  25                                                   PCR03A*       7351-7390  40                                                   DL80-1*       7131-7210  80                                                   DL80-2**      7131-7210  80                                                   DL80-3*       7551-7630  80                                                   DL80-4**      7551-7630  80                                                   500 (ds)      7131-7630  500                                                  280 (ds)      7351-7630  280                                                  159 (ds)      7472-7630  159                                                  121 (ds)      7351-7471  121                                                  Biotin-121*   7351-7471  121                                                  121-.sup.32 P**                                                                             7351-7471  121                                                  79 (ds)       7552-7630  79                                                   Biotin-79*    7552-7630  79                                                   ______________________________________                                         *5' strand.                                                                   **Opposite strand.                                                       

                  TABLE 2                                                         ______________________________________                                        BEAD CAPTURE OF PROBE:TARGET HYBRIDS SHOWS                                    THAT THE RecA-CATALYZED DOUBLE D-LOOP PRODUCT                                 CONTAINS TWO DNA PROBE STRANDS                                                                      .sup.32 P Radioactive DNA                                       Probe Strand(s)                                                                             Counts Captured                                                                .sup.32 P (Radio-                                                                      % Total                                                                              Corrected                                            Biotin   active   Counts per                                                                           % of                                                 (Capture Reporter Minute Counts per                             Reaction                                                                             RecA   DNA)     DNA)     Expected*                                                                            Minute*                                ______________________________________                                        1      -      +        +        4      0                                      2      +      -        +        11     0                                      3      +      +        -        0      0                                      4      +      +        +        110    98                                     ______________________________________                                         *See FIG. 8 for explanation of sample reactions. Percentages were             calculated from the .sup.32 P counts remaining on the DYNAL ™              streptavidincoated beads after three washes of each capture reaction with     1X acetate reaction buffer. The total expected .sup.32 P counts per minut     were determined by scintillation counting of DNA in minced gel slices fro     experiments identical to those in FIG. 8. Since no radioactive DNA was        added to reaction 3 (containing biotin capture probe only), no radioactiv     counts were expected for this reaction.                                       †Radioactive .sup.32 P counts in DNA were corrected for nonspecifi     bead capture of background DNA counts (i.e., counts from reaction 2           without biotin capture probe).                                           

Primers PCR01 and PCR02 correspond to the primers supplied in the"GENEAMP™" DNA Amplification Reagent Kit (Perkin Elmer Cetus, NorwalkConn.).

Single-stranded primers (PCR01, PCR02 and PCR03A) and single-strandedprobes (DL80-1 through 4, biotin-121, 121-³² P, and biotin-79) werechemically synthesized using commercially available phosphoramiditeprecursors on an Applied Biosystems 380B DNA synthesizer (AppliedBiosystems, Foster City Calif.).

DNA molecules were biotinylated by reaction with a biotinphosphoramidite at the last 5' base (New England Nuclear-DuPont, Boston,Mass.) before deblocking. All chemically synthesized DNA molecules weredeblocked according to the manufacturer's specifications.

Short DNA probes (25-mers) were used without further purification.Single-stranded 80-mer and 121-mer DNA probes were purified bypolyacrylamide gel electrophoresis using 8%, and 5% or 8% polyacrylamidegels, respectively. Full sized DNA products were obtained by excisingDNA bands from the gels that corresponded to the correct size DNAmolecule. The DNA molecules were recovered from gel pieces byelectroelution using the "ELUTRAP™" system (Schleicher and Schuell,Keene, N.H.). Both probes and primers were concentrated by standardethanol precipitation (Maniatis et al.). Probe and primer DNAconcentrations were determined based on UV absorbance at 260nm of thediluted DNA.

Double-stranded 500 and 280 bp regions of the lambda genome (FIG. 1)were synthesized using primers PCR01 and PCR02, or PCR03A and PCR02,respectively, Taq polymerase and standard DNA amplification reactionconditions (Perkin Elmer Cetus; Mullis; Mullis et al.). Theamplification products were separated from the DNA primers byelectrophoresis through a 0.7% agarose (Sigma Type II, Sigma, St. LouisMo.) gel (500-mer) or a 4% "NUSIEV™E" (FMC BioProducts, Rockland, Me.)agarose gel (280-mer). The DNA molecules in the bands corresponding tothe amplification products were electroeluted, concentrated, and theiractual concentrations' determined as described above.

Double-stranded 121- and 159-mer probes were obtained by restrictiondigestion of purified 280-mer using the enzyme AluI (New EnglandBiolabs, Beverly Mass.). The DNA probes were isolated by gelelectrophoresis, electroeluted and concentrated as above.

Double-stranded 79-mer probe was obtained from restriction digestion ofthe purified 500-enzyme Hp the enzyme HpaII. The digestion products wereseparated and purified from uncut DNA by electrophoresis using either 3%or 4% "NUSIEVE" (FMC Bioproducts) gels or 1% agarose gels. Specific DNAfragments were recovered from the gels as described above.

Single-stranded or double-stranded DNA molecules were 5'-end-labeled(Maniatis et al.) with γ-³² P!ATP and T4 polynucleotide kinase (Promega,Madison Wis.). When necessary, the DNA molecules were dephosphorylatedwith alkaline phosphatase (Boehringer Mannheim, Indianapolis Ind.)before labelling with T4 polynucleotide kinase. Un-incorporated labelwas removed using "NENSORB 20" nucleic acid purification columns(NEN-DuPont). The labeled DNA molecules could be further purified bydialysis against sterile double-distilled water followed byconcentration by freeze-drying. ³² P-labeled 121-, 159- or 79-mer werealso obtained by the appropriate restriction enzyme digestion of ³² Pend-labeled 280-mer or 500-mer.

EXAMPLE 2 Purification of the Wild-Type RecA and Mutant RecA 803Proteins

RecA and recA-803 proteins were isolated from the overproducing strainsJC12772 and JC15369 (obtained from A. J. Clark and M. Madiraju). Thesestrains contain the RecA and recA-803 coding sequences on plasmidspresent at high copy numbers per cell. Analysis of total protein fromJC12772 and JC15369 cell extracts by SDS-polyacrylamide gelelectrophoresis, under denaturing conditions, showed that the38,000-dalton RecA or recA-803 protein is the major protein produced inthese strains.

RecA and recA-803 proteins were purified by modification of establishedprocedures (Shibata et al., 1981; Griffith et al., 1985) using fastprotein liquid chromatography (FPLC) using a hydroxylapatite columnobtained as a powder (BioRad) followed by an anion ("MONO Q™",Pharmacia) exchange column.

Protein purification was monitored as follows:

(i) identifying the 38,000-dalton RecA protein by SDS-PAGE ("PHASTGEL™"system, Pharmacia, Piscataway N.J.);

(ii) assay of the RecA ssDNA-dependent ATPase activity using γ-³² P!ATPand single-stranded DNA (Shibata et al., 1981). The products of thereaction were separated using PEI cellulose thin-layer chromatography(EM Science, N.J.): the PEI plates were developed in a solvent of 0.5MLiCl and 0.25M formic acid. Products were detected by autoradiography.

(iii) assay of DNase activity. DNase activity was monitored byincubating the RecA protein samples with a mixture of phiX174 linearizedand super-coiled circular double-stranded RF, and circularsingle-stranded DNAs in RecA strand-transfer buffer (Cheng et al., 1988)for 1 hr at 37° C. DNA nicking and digestion were monitored afterdeproteinization by visualizing the DNAs with ethidium bromide afteragarose gel electrophoresis and comparing the quantities of each DNAtype in the RecA incubated samples with those incubated in bufferwithout RecA. Only RecA protein samples showing no detectable DNaseactivity were used.

(iv) assay of D-loop activity with 500-mer oligonucleotide probe using amethod modified from Cheng et al. (1988).

Silver stained SDS-polyacrylamide gel profiles of the final"MONO-Q"-purified RecA and recA-803 proteins showed a single38,000-dalton band from each preparation that was essentially free ofother cellular polypeptides.

EXAMPLE 3 RecA Protein Coating Reactions

RecA protein coating of probes was normally carried out in a standard 1×RecA coating reaction buffer (10× RecA reaction buffer: 100 mM Trisacetate (pH 7.5 at 37° C.), 20 mM magnesium acetate, 500 mM sodiumacetate, 10 mM DTT and 50% glycerol (Cheng et al. 1988). All of theprobes, whether double-stranded or single-stranded, were denaturedbefore use by heating to 95°-100° C. for five minutes, placed on ice forone minute, and subjected in a Tomy centrifuge to centrifugation (10,000rpm) at 0° C. for approximately 20 seconds. Denatured probes were addedimmediately to room temperature RecA coating reaction buffer mixed withATPγS and diluent (double-distilled H₂ O), as necessary.

This reaction mixture typically contained the following components: (i)2.4 mM ATPγS; and (ii) between 10-40 ng of double-stranded probe. Tothis mixture either (i) one μl of RecA protein, usually at 5.2 mg/ml(purchased from Pharmacia or purified as described above), or (ii) anequivalant volume of RecA storage buffer (20 mM Tris-HCl pH 7.5, 0.1 mMEDTA, 1.0 mM DTT, and 50% glycerol) was rapidly added and mixed. Thefinal reaction volume for RecA coating of probe was usually about 10 μl.RecA coating of probe was initiated by incubating probe-RecA mixtures at37° C. for 10 min.

RecA protein concentrations in coating reactions varied depending uponprobe size and the amount of added probe: RecA protein concentration wastypically in the range of 6.8 to 51 μM. When single-stranded DNA probeswere coated with RecA, independently of their complementary probestrands, the concentrations of ATPγS and RecA protein were each reducedto one-half of the concentrations used with double-stranded probes: thatis, the RecA protein and ATPγS concentration ratios were kept constantfor a given concentration of individual probe strands.

FIG. 3 shows an autoradiogram illustrating RecA protein binding to121-mer and 159-mer DNA probes as measured by DNA band-shift assays.Heat denatured ³² P-labeled double-stranded 121-mer and 159-mer DNAprobes were reacted with RecA protein as described above. The finalRecA-DNA reaction mixtures contained 2.4 mM ATPγS. RecA protein or RecAstorage buffer was added to each of four reactions containing 0.01 μg ofeither denatured 121- or 159-mer DNA probe. The final concentration ofRecA in each reaction was 0, 0.137, 1.37 or 13.7 μM in lanes 1 and 5, 2and 6, 3 and 7 or 4 and 8, respectively (FIG. 3). All RecA/DNA probecoating reactions were performed in a final volume of 10 μl. RecAbinding was initiated by incubating all the reactions at 37° C. for 10min. Five μl aliquots of each reaction were loaded into a 2% agarose gelin 1× TBE buffer and electrophoresed at 9.2 v/cm for 2 hours. A HaeIIIdigest of φX-174 DNA (GIBCO-BRL, Gaithersburg Md.) served as adouble-stranded DNA size marker (M). Marker DNA was 5' end-labeled with³² P as described above. The gel was air dried on saran wrap in aBioscycler oven at 65° C. The dried gel was then exposed to X-ray film.

As can be seen from FIG. 3, retardation of the electrophoretic mobilityof the DNA probes increases with increasing RecA concentration.

The same conditions as described above were employed for recA-803protein.

EXAMPLE 4 Formation of RecA Protein-Mediated Multiplexes

Probe coating reactions were performed as described in Example 3. Afterthe coating reactions were complete target DNA was added to eachreaction. The target DNA was derived from the lambda viral genome and ifrestriction enzyme digested, contained DNA fragments homologous to theprobe sequence and non homologous ones as well. Typically, 0.66-1.5 μgof target DNA was added to each reaction in 1× reaction buffer. Themagnesium ion concentration of the total reaction was adjusted to 12 mMby addition of an aliquot of 0.2M magnesium acetate. Final reactionvolumes were usually 20 μl after the addition of the target DNA.

Probe target mixtures were incubated at 37° C. to allow RecA catalyzedhomologous probe:target reactions. After incubation for 60 minutes thereactions were deproteinized with proteinase K (10 mg/ml) at 37° C. for15-20 min, followed by the addition of sodium dodecylsulfate (SDS) to afinal concentration of 0.5-1.2% (w/v). Aliquots of each reaction wereloaded into wells of 0.7-1.0% agarose gels after addition of trackingdye (Maniatis et al.). The gels were electrophoresed either at roomtemperature or at 4° C. The gels were stained with ethidium bromide andDNA molecules visualized by UV light. The gels were photographed using ared filter and Polaroid 667 black and white film.

When radiolabeled DNA probes were utilized in the reactions, theprobe:target complexes were detected by autoradiography of either wet ordried agarose gels using either DuPont CRONEX QUANTA III™ or LIGHTENINGPLUS™ intensifying screens and Kodak X-OMAT AR5™ film. For signalquantitation, target DNA bands showing signal on autoradiograms wereexcised from gels, crushed, suspended in scintillation cocktail(AQUASOL-2™, DuPont-NEN, Boston Mass.), and the radioactivity counted ina Packard 2000 CA Tri-Carb liquid scintillation analyzer.

Double-stranded 280-mer and 500-mer probes (Table 1) were reacted with adouble-stranded linear target DNA fragment (8370 bp lambda DraI digestfragment that contains each probe sequence) as described above.Denatured probe DNA molecules were coated with RecA protein in thepresence of 2.4 mMATPγS and 34.2 μM RecA protein. Denatured probes thatwere not coated with RecA protein were added to the same reactionconditions minus the RecA protein. After incubation for 10 min at 37°C., 0.66 μg of lambda genomic DNA, which had been digested with therestriction enzyme DraI, was added to each probe mixture: the genomicDNA was suspended in 1× reaction buffer with an adjusted Mg⁺⁺concentration, as described above. The final micromolar ratio ofdouble-stranded probe to homologous double-stranded target fragment was10.6:1 for 500-mer and 21.6:1 for 280-mer. Incubation of theprobe:target reactions were carried out as described above.

The reactions were deproteinized and loaded into a 0.7% agarose gel. TheDNA was subjected to electrophoresis to separate the probe and targetDNA fragments. A photograph of the ethidium bromide stained DNA in thegel containing these reactions is shown in FIG. 4A. The three reactionson the left side are control reactions using pUC18 double-strandedcircular DNA target and RecA coated single-stranded 69-mer probe (thesecontrol substrates were provided by B. Johnston, SRI, Menlo Park,Calif.). The center lane contained 1 kb marker DNA (GIBCO-BRL). The fourreactions on the right side of the gel were the lambda DraI digesttarget DNAs.

This gel was dried and exposed to X-ray film as described above. Theresulting autoradiogram is shown in FIG. 4B. Both RecA coated 500-merand 280-mer probes specifically hybridized with the correct DraI lambdaDNA digest target fragment. The position of probe:target DNA homology isat least 832 bp from the 3' end of the 8370 bp target fragment. Thisresult demonstrates the formation of 500-mer and 280-merRecA-facilitated DNA hybridization products that are stable todeproteinization. Generation of these stable DNA complexes requires RecAprotein.

EXAMPLE 5 Stable Complex Formation Between Small Double-Stranded Probesand Linear Double-Stranded Target DNAs

This example describes the use of small double-stranded probes togenerate complexes with linear double-stranded DNA that are stable todeproteinization.

The following hybridization reactions were carried out as described inExample 4. All RecA protein-coating reaction volumes were 10 μl. Eachreaction contained 2.4 mM ATPγS and 20.5 μM RecA, unless otherwisenoted. Final reactions contained 1.3 μg DraI digested lambda DNA. Thefollowing reaction conditions correspond to lanes in FIGS. 5A and 5B:Lanes 1 and 2, 280-mer probe with and without RecA protein,respectively; lane 3, 121-mer probe with RecA protein; lanes 4-6, 79-merprobe with RecA protein. The reactions in lanes 5 and 6 contained RecAprotein concentrations of 8.54 and 41 μM during the RecA probe coatingreaction. The reactions were deproteinized and loaded into a 0.7%agarose gel. The gel was subjected to electrophoresis to separate theprobe and target DNA fragments.

FIG. 5A shows a photograph of ethidium bromide stained DNA in an agarosegel showing the lambda DraI digest target fragments from the abovereactions. FIG. 5B shows an autoradiograph of the DNA in an agarose gelshown in FIG. 5A. The arrows indicate the migration position of the DraIlambda target fragment homologous to the probes used. The resultsindicate that complexes stable to deproteinization can be achieved withall of the RecA-coated DNA probes: 280-mer, 121-mer, and 79-mer. Asabove, the stable complexes were formed at least 832 bases from the endof a linear double-stranded DNA target molecule.

EXAMPLE 6 Stable Complex Formation Requires a Double-Stranded Probe

A. Requirement for a Double-Stranded DNA Probe.

RecA-coated DNA probes were used for hybridization with 3.0 μg of lambdaDraI digested target DNA per reaction (60 minutes) as described inExample 4. Two chemically synthesized single-stranded DNA 121-mers wereused. The strands were complementary to each other. One strand wasbiotin-labeled and the other ³² P-labeled.

The following reaction numbers correspond to lanes in FIGS. 6A and 6B.Reactions 1, 2, 5, 6 and 3, 4 contained 2.4 mM or 4.8 mM ATPγS,respectively. All reactions used 20 ng of the ³² P-labeled DNA probestrand and 10.4 μg RecA protein: each reaction contained the same ³²P-specific activity. Reactions 3-6 also contained the biotin labeledstrand. In reactions 3 and 4, both DNA probes were added to the initial10 μl RecA reactions at the same time. For reactions 5 and 6, the ³² P-and biotin-labeled probes were each coated with RecA in separate 10 μlreactions, then one-half of each reaction mix was incubated with targetDNA for 30 min before addition of the missing complementary RecA coatedDNA probe strand. The ³² P-labeled DNA strand was added first toreaction 5 and second to reaction 6.

All reactions were deproteinized with proteinase K and SDS beforeelectrophoresis. FIG. 6A shows a photograph of ethidium bromide stainedDNA in an agarose gel showing the lambda DraI digest target fragmentsfrom the above reactions, The reaction conditions are summarized inFIGS. 6A and 6B. The autoradiogram of the air dried gel of FIG. 6A isshown in FIG. 6B.

Two DNA probe strands are required for the production of stabledeproteinized products formed at a homologous internal sequence onlinear target DNA (lanes 3, 5, and 6, compare lane 2). RecA protein isalso required for product formation (lanes 3, 5 and 6, compare lane 4).

B. A Model of the Double-Stranded Stable Product.

FIG. 7 shows a possible model for the deproteinized double-stranded RecAcatalyzed hybridization product. FIG. 7 illustrates a stable doubleD-loop mutliplex formed with short end-labled DNA probes and adouble-stranded linear DNA target. The model shown depicts hybridizationon the 8370 bp DraI DNA lambda target, where probe:target homologybegins at least 832 bases from the short end of the target (3' end withrespect to the whole lambda genome). The DraI fragment includes lambdabases 93-8462. The exact regions of homology are defined by Table 1. Asingle-stranded RecA protein coated probe does not yield complexes thatare stable to deproteinization (see FIG. 6B above).

EXAMPLE 7 Probe:Target Capture and DNA Detection

A. Hybridization Reactions.

Complementary 121-mer DNA probes (Table 1) were individually chemicallysynthesized. The complementary strands were differentially labeled usingγ-³² P!ATP and biotin (the reporter and capture moieties, respectively).All probe coating reaction mixes contained 2.4 ,μM ATPγS, 20 ngsingle-stranded probe, 5.2 μg of RecA protein (5.2 μg/μl; Pharmacia), oran equivalent to volume of RecA storage buffer (without RecA protein),per 10 μl reaction. For experiments using both biotin- and ³² P-labeledprobes (reactions 1 and 4) , 10 μl aliquots of the analogous biotin- or³² P-labeled probe-coating reactions were mixed together (to give 20 μl)before these mixtures were added to the target DNA mix. To keep allreaction volumes constant, reactions with only one single-stranded probestrand (reactions 2 and 3) used 10 μl of probe mix, 10 μl of probereaction buffer (i.e., no second probe) and 20 μl of target DNA mix.

A lambda target DNA mix was prepared as follows: 1× RecA reactionbuffer, 1 to 10 dilution of 0.2M stock Mg acetate, and ApaI digestedlambda DNA. Twenty microliters of the target DNA mix was added to eachof the 20 μl probe reaction mixtures. These reactions were incubated for60 minutes at 37° C. The 40 μl reactions were deproteinized, dividedinto two equal aliquots, the two aliquots loaded in adjacent wells in a0.7% agarose gel and the components fractionated by electrophoresis (asdescribed above).

The initial specific activity of all reactions containing the ³²P-labeled strand (1, 2 and 4) were identical. The ethidium bromidestained gel with adjacent duplicate lanes of each reaction is shown inFIG. 8. The contents of each reaction are summarized in FIG. 8.

The portion of each lane of the gel corresponding to the 10.1 kb lambdatarget DNA (FIG. 8) was excised from the gel. Each excised fragment wasplaced into a microcentrifuge tube and rapidly frozen using dry ice. TheDNA contained in each gel fragment was recovered by squeezing the frozengel between folded parafilm until no more liquid was extruded. ThisDNA-containing liquid was then carefully removed with an EPPENDORF™micropipette.

B. The Capture/Detection Assay.

The presence of two probe strands on the same target molecule (onebiotin-labeled and the other ³² P- labeled) was assayed by capturingbiotin-containing-probe:target hybrids on streptavidin-coatedparemagnetic beads (Dynal, Oslo, Norway). The manufacturer's beadstorage buffer was removed before use. The beads were washed in 1× RecAreaction buffer, in 10× RecA reaction buffer, and finally in RecAreaction buffer. Before DNA capture, equal aliquots of washed beads wereadded to individual 1.5 ml microcentrifuge tubes and the final washbuffer was removed. Liquid was removed from all bead suspensions byplacing microcentrifuge tubes containing the bead mixtures in a magneticseparating rack (Promega, Madison Wis.).

The DNA-containing reaction samples from above were each added to amicrocentrifuge tube containing an aliquot of the washed paremagneticbeads. The samples were mixed, and incubated at room temperature for 15min. Since beads settle with time, the mixtures were shaken severaltimes during incubation to insure efficient biotin:streptavidinexposure. After the capture reaction, i.e., the binding of streptavidinto biotin, the paramagnetic beads in each reaction were amassed with amagnet and the reaction liquid removed.

Each sample of beads was washed three times with 1× RecA reactionbuffer. The presence of ³² P-labeled probe strand was assessed by addingliquid scintillation counting fluor to the beads and counting theradioactivity of the DNA captured by each bead reaction. These data arepresented in Table 2.

                  TABLE 2                                                         ______________________________________                                        BEAD CAPTURE OF PROBE:TARGET HYBRIDS SHOWS                                    THAT THE RecA-CATALYZED DOUBLE D-LOOP PRODUCT                                 CONTAINS TWO DNA PROBE STRANDS                                                                      .sup.32 P Radioactive DNA                                       Probe Strand(s)                                                                             Counts Captured                                                                .sup.32 P (Radio-                                                                      % Total                                                                              Corrected                                            Biotin   active   Counts per                                                                           % of                                                 (Capture Reporter Minute Counts per                             Reaction                                                                             RecA   DNA)     DNA)     Expected*                                                                            Minute.sup.3                           ______________________________________                                        1      -      +        +        4      0                                      2      +      -        +        11     0                                      3      +      +        -        0      0                                      4      +      +        +        110    98                                     ______________________________________                                         *See Figure 8 for explanation of sample reactions. Percentages were           calculated from the .sup.32 P counts remaining on the Dynal ™              streptavidin  coated beads after three washes of each capture reaction        with 1X acetate reaction buffer. The total expected .sup.32 P counts per      minute were determined by scintillation counting of DNA in minced gel         slices from experiments identical to those in Figure 8. Since no              radioactive DNA was added to reaction 3 (containing biotin capture probe      only), no radioactive counts were expected for this reaction.                 †Radioactive .sup.32 P counts in DNA were corrected for nonspecifi     bead capture of background DNA counts (i.e., counts from reaction 2           without biotin capture probe).                                           

In Table 2 percentages were calculated from the ³² P radiolabelled DNAcounts minute remaining on the DYNAL™ streptavidin-coated beads afterthree washes of each capture reaction with 1× acetate reaction buffer.No ³² P-labeled DNA was added to reaction 3, which contains biotincapture probe only: no radioactive DNA counts were expected for thisreaction. The "Total Expected Counts" in Table 2 were determined asfollows. Identical reactions were performed as described above and theproducts separated by agarose gel electrophoresis. Gel fragmentscorresponding to the 10.1 kb target DNA were excised from the gel,minced, and placed in AQUASOL®. The amount of ³² P-labeled DNA presentin the samples was determined by liquid scintillation counting.

The results indicate that the hybridization product, containing twocomplementary but differentially labeled probes, can be captured usingthe streptavidin interaction with the biotin labeled probe strand andsubsequently detected by a label in the complementary probe strand.

This bead capture of stable probe:target hybrids supports thathomologous probe:target complexes catalyzed by RecA protein actuallycontained two homologous probe strands on the same double-strandedtarget molecule.

EXAMPLE 8 RecA+ Facilitated DNA Amplification Without Target DNADenaturation

Reaction conditions for RecA protein facilitated DNA amplification havebeen described in co-pending and co-owned Ser. No. 07/520,321, for"PROCESS FOR NUCLEIC ACID HYBRIDIZATION AND AMPLIFICATION," filed 7 May1990, herein incorporated by reference.

Double-stranded probe/primer pairs corresponding to DL801/2 and DL803/4(Table 1) were denatured and coated with RecA protein as describedabove. To ensure that elongation of DNA primers occurs in only thedesired direction, the 3'-ends of the appropriate primers can beterminated by a 2',3'-dideoxynucleotide. The dideoxynucleotide lacks the3-hydroxyl group present in the conventional dNTPs. The absence of thehydroxyl group inhibits extension by preventing. the formation of aphosphodiaster bond between the dideoxynucleotide and the succeedingconventional dNTP. The addition of the dideoxynucleotide to the primercan be achieved by using the enzyme terminal deoxynucleotide transferase(Pharmacia, Piscataway, N.H.).

The probes are then allowed to react with the target DNA as describedabove. The product of the above reaction, consisting of two sets ofdouble D-loops, is then used as the substrate in a typical DNAamplification reaction. The DNA reaction can be carried out in buffercontaining 10 mM Tris-HCl (pH 7.5), 8-12 mM MgCl₂, and 50 mM NaClsupplemented with 200-750 μM dNTPs and DNA polymerase (e.g.,exonuclease-free, DNA polymerase I, Klenow, or T7 DNA polymerase). Inaddition, the reaction may be supplemented with other enzymes orproteins (e.g. DNA helicase, topoisomerase, DNA ligase and single-strandbinding (SSB) protein) which may facilitate the formation of thespecific amplification product. The reaction is allowed to proceed foras long as necessary at 37° C. Upon termination, samples could bedeproteinized (SDS, Proteinase K and/or phenol extracted) and analyzedby gel electrophoresis. After electrophoretic separation, the resultingamplified DNA can be visualized by either ethidium bromide staining ofthe DNA in the gel or by DNA hybridization with a target specific DNAprobe. Alternatively, amplification DNA probes could be biotinylated andthe newly synthesized DNA captured by appropriate means and thenreported and detected as previously described.

DNA synthesis reactions are initiated by the addition of 1-2 unit(s) ofexonuclease-free E. coli DNA polymerase I (U.S. Biochemicals) and 750 μMof each dNTP. The reactions are maintained at 37° C.

Following the initial addition of polymerase, the reactions can besupplemented with 1 unit of e.g., Klenow and/or additional dNTPs, atspecific intervals spaced over the time course of the reaction.

Samples are treated with proteinase K, before being loaded forelectrophoretic separation. After electrophoretic separation theresulting amplified DNA fragments can be visualized by either ethidiumbromide staining of the gel or by hybridization with a target specificprobe.

For hybridization analysis the gel can be transferred by standardprotocols (Maniatis et al.) onto hybridization transfer membrane(Hybond-N, Amersham). The DNA is UV cross-linked (Stratolinker,Stratagene) to the membrane. The UV-treated transfer membrane ishybridized with end-labelled (Boehringer Mannheim) probe PCR03A (Table1): PCR03A (nucleotides 7351 through 7390 of the native lambda genome)is a 40-mer corresponding to an internal DNA sequence of the 500 basepair lambda template that is the target of the above amplificationreaction. The membrane is subjected to autoradiography.

EXAMPLE 9 In situ DNA Detection Utilizing the Double D-loop Reactions

A. Preparation of Probe Complex.

Biotinylated chromosome X alpha satellite DNA probe is obtained fromONCOR (Gaithersburg, Md.). Alternatively, probes can be biotinylated bystandard nick-translation methods or by polymerase chain reaction (Weieret al., 1990).

The double-stranded probe diluted in sterile ddH₂ O to the desiredconcentration prior to denaturation, is denatured in a 0.5 mlmicrocentrifuge tube in a 100° C. heat block for 5 minutes. The tube isimmediately placed in an ice water bath for 1 to 2 min followed by abrief centrifiguration at 4°-6° C. in a TOMY™ microcentrifuge, and thetubes are returned to an ice water bath. Approximately 5 minutes priorto addition of denatured DNA probe to the hybridization mixture the tubecontaining the probe is placed in ice in a freezer at -20° C. The probehybridization mixture contains the following-components in a broad rangeof concentrations and is combined in the order listed: 1 μl of 10× RecAreaction buffer 10× RecA reaction buffer:100 mM Tris acetate pH 7.5 at37° C., 20 mM magnesium acetate, 500 mM sodium acetate, 10 mM DTT and50% glycerol (Cheng et al., 1988)!; 1.5 μl ATPγS from 16.2 mM stock)(Pharmacia) GTPγS, rATP (alone or in the presence of a rATP regeneratingsystem), dATP, mixes of ATPγS and rATP, or mixes of ATPγS and ADP, mayalso be used in some reactions!; 0.75 μl 20 mM magnesium acetate; 4-60ng (or more in some reactions) of denatured probe in sterile water; 1.25μl 0.137 mM stock RecA protein when purchased from Pharmacia whenobtained from other sources or prepared in the laboratory the amount(μl's) added varies according to concentration of stock!.

The mixture is incubated at 37° C. for 10 minutes followed by additionof 0.5 μl/reaction of 200 mM magnesium acetate. Final concentrations ofreaction components are: 4.0 mM to 10 mM Tris acetate, 2.0 mM to 15 mMmagnesium acetate, 20.0 mM to 50 mM sodium acetate, 0.4 mM to 1.0 mMDTT, 2% to 5% glycerol, 1 mM to 2.5 mM ATPγS, 0.005 mM to 0.02 mM RecAprotein.

B. Preparation of HEp-2 Fixed Cell Nuclei.

HEp-2 cells were originally derived from human male larynx epidermoidcarcinoma tissue. HEp-2 is a chromosome ploidy variable cell line(Chen).

The cells are cultured for 24 hours after seeding in DMEM medium(Whittaker or Gibco-BRL) supplemented with 10% FBS, sodium pyruvate anda penicillin/streptomycin antibiotic mix at 37° C. under standardconditions. Cells are pelleted by low-speed centrifugation and thepellet is resuspended in 75 mM KCl in a 37° C. water bath for between 5and 15 minutes for the desired amount of nuclear swelling to occur,followed by cell fixation accomplished by the addition of 3:1 ice coldmethanol:acetic acid and centrifugation at 6° C.

One ml of fluid is left in the tube with the pelleted cells, additionalice cold methanol:acetic acid is added, and the cells mixed by gentlemixing of the tube, followed by centrifugation. Repeated additions ofmethanol-acetate degrades cytoplasm (HEp-2 and other cell types may befixed in alternative ways, some of which do not degrade cytoplasm.)

Preparations of isolated nuclei are fixed by resuspension in 3:1methanol:acetic acid at a concentration ˜2×10⁶ /ml and is either droppedby pipette in 10 μl aliquots onto clean glass slides which are stroredat -20° C. or the suspended nuclei are stored at -20° C. for later use.

C. Hybridization Reactions for Fixed Preparations.

Ten μl of probe mixture/reaction from Example 9A is applied to the fixedpreparation on glass slides. Glass coverslips are placed over thehybridization areas and sealed with rubber cement, and reactions areincubated in a moist container in a 37° incubator for between 1-4 hours.

Following incubation, the rubber cement is manually removed and theslides are washed in coplin jars 3 times for 10 minutes each in 2× SSC(20× SSC: 3NaCl, 0.3M sodium citrate, pH 7.0 is used in all SSCcontaining preparations in these assays) in a water bath at 37° C. Otherwashing conditions may also be employed.

The slides are placed in pro-block solution 4× SSC, 0.1% TRITON®X-100,5% Carnation nonfat dry milk, 2% normal goat serum (Gibco), 0.02% sodiumazide, pH 7.0! for 25 minutes at room temperature (RT), followed byimmersion in 5 ug/ml FITC-avidin DCS cell sorter grades (Vector, A-2011)in preblock solution for 25 minutes at room temperature. The slides aresuccessively washed in 4× SSC, 4× SSC and 0.1% TRITON®X-100, and 4× SSCfor 10 minutes each at room temperature, followed by brief rinsing indouble-distilled water. The slides are then dried.

Antifade solution is applied to the slides 100 mg p-phenylenediaminedihydrochloride (Sigma P1519) in 10 ml phospate buffered saline,adjusted to pH 8 with 0.5M carbonate-bicarbonate buffer (0.42 g NaHCO₃adjusted to pH 9 with NaOH in 10 ml dd₂ O) added 90 ml glycerol, and0.22 um filtered!, and coverslips are placed over the preparations.Anitifade containing a counterstain such as propidium iodide or DAPIsolution can be used instead of anitifade alone.

If necessary, signal amplification is performed as follows: Slides arewashed for 5-10 minutes in 4× SSC and 0.1% TRITON®X-100 at RT to removecoverslips and antifade, followed by incubation in preblock solution forup to 20 minutes. The slides are then incubated with biotinylated goatanti-avidin antibody (Vector BA-0300) at a concentration of 5 ug/mldiluted in pre-block solution for 30 minutes at 3720 C. Slides aresuccessively washed for 10 minutes each in 4× SSC, 4× SSC and 0.1%TRITON® X-100 4× SSC at RT, then immersed in preblock solution for 20minutes at RT, then immersed in preblock solution with 5 ug/mlFITC-avidin for 20 minutes at RT. Slides are again washed in the 4× SSCseries, briefly rinsed in ddH₂ O, and on slides mounted with an antifadeor antifade with counterstain.

Specific signals detected using standard fluorescence microscopyobservation techniques.

D. Dectection of Specific Chromosome Sequences in Fixed Nuclei and WholeCells.

The hybridization mixture is combined in the following order: 1 μl 10×RecA reaction buffer, 1.5 μl ATPγS (16.2 mM stock, Pharmacia), 0.75 μlmagnesium acetate (20 mM stock), 12 μl (Example 8A) containing 20 to 60or more ng of danatured probe in ddH₂ O, RecA (0.137 mM stock,Pharmacia). The mixture is incubatad in a 37° C. water bath for 10minutes followed by addition of 0.5 μl 200 mM magnesium acetate.

EXAMPLE 10 RecA Mediated Double D-loop Hybridization Reactions Using aVariety of Cofactors

This example describes the formation of the double-D-loop complex usinga number of different cofactors for the RecA protein coating reactions.

Double-D-loop reactions were carried out in 1× D-loop buffer (10×buffer: 100 mM Tris acetate (pH 7.5 at 37° C.), 20 mM magnesium acetate,500 mM sodium acetate, 10 mM DTT and 50% glycerol (Cheng et al. 1988))using 38 ng probe and containing ATPγS (Pharmacia), rATP (Pharmacia),dATP (USB) or GTPγS (Pharmacia) at a concentration of 1.2 mM. Thereactions were established with or without a regenerating system andcontained 1.2 μg λ/ApaI target DNA digest (New England Biolabs, BeverlyMass.). The probe was the Lambda 280-mer (Table 1) end-labeled with ³² P(Ausubel, et al.). The Lambda target fragment, i.e., the ApaI fragmentcontaining sequences homologous to the probes, is the smaller 10.1 kbfragment indicated by an arrow in FIG. 16. The final concentration ofRecA in all RecA containing reactions was 12.3 mM. Typically, the finalmagnesium acetate concentration was approximately 12 mM in each reaction(Example 4).

The double-D-loop formation reactions were deproteinized using 10 mg/mlproteinase K and 0.5% SDS. The deproteinized RecA mediated double D-loophybridization reactions containing heat denatured 280-mer probe anddifferent cofactors described above, were resolved by electrophoresis onan agarose gel. The gel was stained with ethidium bromide (Maniatis, etal.) and a photograph of the gel is shown as FIG. 16. The gel was driedand exposed to X-ray film.

FIG. 17 is an autoradiograph of the dried gel shown in FIG. 16. In FIG.17, the lanes correspond to the following reaction conditions. RecA:lane 2, no RecA; lanes 1, 3-7, +RecA. Cofactors: ATPγS, lanes 1 and 2;rATP, lanes 3 and 4; dATP, lanes 5 and 6; GTPγS, lane 7. Lanes 3 and 5also contained an ATP regenerating system 6 mM creatine phosphate, 10U/ml phosphocreatine kinase (Sigma, St. Louis Mo.) and 100 mg/ml BSA(Promega, Madison Wis.)!. All reaction conditions were as describedabove.

The sample origin is indicated in FIGS. 16 and 17. As can be seen fromthe results presented in FIG. 17, stable double D-loop complexes wereformed in the presence of each cofactor, as indicated by the labeledbands corresponding to the location of the 10.1 kb lambda targetfragment (arrow).

EXAMPLE 1 RecA Mediated Double D-Loop Hybridization Reactions ContainingATPγS or a Mixture of ATPγS and rATP

This example describes the formation of the double-D-loop complex usingATPγS and mixtures of ATPγS/rATP as cofactors for the RecA proteincoating reactions.

The reaction conditions were as described in Example 10. FIG. 18 showsthe photograph of an ethidium bromide stained agarose gel on whichcomponents of deproteinized RecA mediated double D-loop hybridizationreactions, using 20 ng heat denatured 500-mer probes (Table 1), wereresolved by electrophoresis. The probes were end-labeled with ³² P asabove. The gel was dried and exposed to X-ray film.

FIG. 19 shows an autoradiograph of the dried gel in FIG. 18. In FIG. 19,the lanes correspond to the following reaction conditions. Lanes 1, 3and 5--ATPγS cofactor (1.2 mM). Lanes 2, 4 and 6--a combination of ATPγSand rATP cofactors (0.73 mM and 0.5 mM, respectively). Lanes 1, 2 and 3,4--reactions done with two different lots of λ/ApaI target DNA digest(New England Biolabs). Reactions in lanes 5 and 6 used a λ/DraI targetDNA digest. 7A reactions contained 3.5 μg of target DNA mixture. RecAconcentrations in all reactions were 6.85 μM. All concentrations arebased on a final volume of 20 μl. All reaction conditions were as above.

The sample origin is indicated in FIGS. 18 and 19. As can be seen fromthe results presented in FIG. 19, stable double D-loop complex wereformed in the presence of each cofactor, as indicated by the labeledbands corresponding to the location of the 10.1 kb and 8.4 kb lambdatarget fragments (arrows).

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

EXAMPLE 2 A homogeneous diagnostic assay

A. Labeling of lambda DNA

To facilitate evaluation of the capture reaction, lambda DNA (BRL),heated to 65° C. for 5 min, was end-labeled with ³² P label using aKlenow fill-in reaction. The labeling reaction contained 10 μl 10×Klenow buffer (50 mM Tris HCl pH 7.5, 5 mM MgCl₂, 10 mMβ-mercapto-ethanol) 18.3 μl 0.82 μg/μl lambda DNA, 2 μl 5 U/μl KlenowDNA polymerase (Pharmacia) and 56.7 μl dd H₂ O. After incubation a 37°C. for 30 min the reaction was spun through a SEPHADEX® G-50 (Pharmacia)column in a 1 ml syringe, Labeled DNA was precipitated in ethanol in thepresence of 0.3M NaOAc, resuspended in 20 mM Tris-HCl pH 7.5, 0.1 mMEDTA (TE) and then ethanol precipitated a second time. The dried DNAconcentration was resuspended in 45 μl TE buffer. The DNA concentrationwas determined by its absorbance at 260 nm in a spectrophotometer.

B. DNA Probe synthesis and biotinylation

A 1000 bp region of the lambda genome was synthesized using standardprotocols for thermally cycled PCR. The reaction used two chemicallysynthesized primers PCRO2 and PCR01000 SEQ ID NO: 13 including bases6631 to 6655 on the lambda genome), all four dNTP precursors and Taq DNApolymerase (Promega). Synthesized 1000-mer DNA (including lambda bases6631 to 7630) was centrifuged through a Sephadex G-50 (Pharmacia) columnand the DNA recovered by ethanol precipitation (2×). The 1000-mer DNAwas resuspended in TE buffer, and its concentration was determined by ODmeasurement at 260 nm and varified with the DNA DIPSTICK™ (Invitrogen).Purified 1000-mer was then labeled with bio-14-dATP (Gibco-BRL) usingthe BRL, Nick Translation System. By slightly modifying the BRL protocoland adding twice the recommended amount of enzyme mix and incubating at15°-16° C. for 1 hr 15 min, DNA probes with an average single-strandsize of 300-500 bases were obtained. Nick-translated probes wereprecipitated in 0.3M NaOAc in ethanol and after resuspension in TE, DNAconcentration was determined with the DNA Dipstic™ (Invitrogen).

C. RecA-coating of probes and probe:target hybridization

The single-stranded nick-translated probe was coated with RecA proteinin a reaction mixture containing 1 μl of 10× acetate reaction buffer(Cheng et al, 1988) 1.5 μl of 3.24 mM ATP.sub.γ S (Sigma) 0.6 μl of recA(2.76 μg/μl), 4.4 μl of sterile ddH₂ O, and 2 μl of heat denatured DNAprobe (15 ng/μl). The DNA probe was heat denatured at 100° C. for 5 min,quick-cooled in an ice-water bath, centrifuged at 4° C. in a Tomymicrocentrifuge for 20 sec to collect the liquid, and then the properaliquot was immediately added to a mixture containing the other reactioncomponents. The total volume of the RecA-coating reaction mix afterprobe addition was 10 μl. The probe mix was incubated at of eachcofactor, as indicated by the labeled bands 37° C. for 15 min followedby addition of 10 μl of a lambda target DNA mix containing 4 μl of 10×acetate reaction buffer, 4 μl of 0.2M Mg(OAC)₂, 4 μl of ³² P-labeledwhole duplex lambda DNA (280 ng/μl; previously heated at 65° C. for 5min) and 28 μl of ddH₂ O. The RecA-mediated hybridization reaction wasincubated at 37° C. for 60 min, then 1.3 μl of 300 mM EDTA (pH 8.0) wasadded to give a final concentration of -20 mM. This was followed byaddition of 21 μl of 20 mM Tris-acetate buffer pH 7.5 with 1M NaCl, togive a final salt concentration of 0.5M. Three control reactions wererun along with the RecA reaction containing lambda probe. The firstcontrol reaction was identical to the RecA reaction except that it didnot contain RecA protein and instead, an equivalent amount of RecAstorage buffer was added. The two other control reactions were identicalto the experiments containing lambda probes except that the lambdaprobes were replaced by an equivalent amount of nonlabeled,nick-translated φx174 DNA RFI DNA (NEBiolabs). Approximately 5 minbefore use, 300 ml of DYNABEADS® (Dynal) were washed three times in 20mM Tris-acetate pH 7.5, 1M NaCl. Wash buffer was removed by amassing thebeads in a magnetic separating rack (Promega) and each hybridinationreaction (in 0.5M NaCl) was added to a separate aliquot (100 μl) ofwashed beads in a microcentrifuge tube and incubated with beads at roomtemperature for 30 min, with occasional gentle shaking of the tubes tocarefully resuspend the beads in the reaction liquid. After capture, theliquid was removed and the beads were washed 3× with 100 μl 20 mMTris-acetate pH 7.5, 1M NaCl.

The radioactivity on the beads and in the washes was determined bycounting in SAFETY-SOLVE™ (RPI Corp.) in a Packard Scintillationcounter. The results of these experiments are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        The double D-loop hybridization reaction with                                 RecA-coated biotinylated complementary lambda DNA                             probes allows the specific capture of double-stranded                         -50 kb lambda viral target DNA on magnetic beads.                                                            % Capture of                                              Single-             .sup.32 P-Labeled                                         stranded    RecA    Lambda                                         Reaction   Probe DNA   Protein DNA Target                                     ______________________________________                                        1          lambda      +       45.9                                           2          lambda      -       3.7                                            3          φX174.sup.a                                                                           +       3.6                                            4          φX174.sup.a                                                                           -       3.1                                            ______________________________________                                         .sup.a Non-biotinylated, nicktranslated RFI                              

Legend to Table 3: RecA-mediated double D-loop reactions usingbiotinylated (nick-translated with bio-4-dATP) lambda DNA probes, ornon-labeled (nick-translated with dATP) φX174 RFI probes, and ³²P-labeled whole lambda genomic DNA targets were carried out. Thesingle-stranded probes obtained by nick-translation averaged 300-500bases in size and the lambda DNA probes were all homologous to acontiguous 1000 bp region of the lambda viral genome. RecA-coated ssprobes were reacted with lambda target DNA for 1 hr at 37° C., treatedwith 20 mM EDTA and 0.5M NaCl, and affinity captured on freshly washedstreptavidin-coated magnetic DYNABEADS® (Dynal). Non-captured DNA wasremoved from the reaction mixture by washing. The % of ³² P-labeledlambda DNA remaining on the beads after washing was determined byscintillation counting. Reactions without RecA protein and/or with φX174DNA sequences as probe, served as controls (see Text and Methods fordetails). The results show that double D-loop hybrids formed betweennick-translated probes and large double-stranded Target DNAs can bespecifically captured and detected with magnetic beads.

D. Signal amplification

For The purpose of detecting captured target DNA when the target is notlabeled and/or for amplifying signal for detecting low copy numbertargets, the washed Dynabeads® with attached captured DNA, were washedonce in 1× T7 buffer (10× buffer: 400 mM Tris HCl pH 7.5, 100 mM MgCl₂,50 mM DTT) and then resuspended in 44 μl of amplification reaction mixcontaining 31.1 μl ddH₂ O, 0.5 μl each of 100 mM dCTP, dGTP and dTTP,9.4 μl 0.53 mM bio-14-dATP (BRL) and 2 μl 0.8 μM of poly(A) primer SEQID NO.4 included bases (without poly (A) tail), are numbers 8001 to 8023on the lambda genome. The primer, target DNA mixture was then heated to00° C. for 5 min and cooled to -37° C. for -10 min before the followingwas added; 5 μl 10× T7 buffer, 0.5 μl 13 U/μl T7 SEQUENASE® Version 2.0(USB), and ddH₂ O to a final volume of 50 μl. The reaction was thenincubated for 1 hr at 37° C. before being stopped by addition of 5 μl of0.3M EDTA pH 8.0.

E. Signal detection

Incorporation of bio-14-dATP into primer-synthesized DNA was detectedusing the SOUTHERN-LIGHT™ (Tropix) chemiluminescence assay. DNA fromamplification reactions with and without T7 enzyme were dilutedappropriately in TE and 1 μl of DNA mix was added to 4 μl 200 mM NaOHwith 12.5 mM EDTA, incubated at room temperature for 5 to 10 min, thenspotted onto dry TROPILON-45™ nylon membrane on plastic wrap. DNA spotswere air dried, the dried membrane was transferred onto 3 MM CHR(Whatman) chromatography paper and 20× SSC was dropped onto the 3 MMpaper around the edges of the nylon. When the DNA dots were wetted, DNAwas crosslinked to the membrane with a Stratagene Stratalinker set on"auto". After the nylon was fully wetted by 20× SSC, the SOUTHERN-LIGHT™(Tropix) biotinylated DNA detection procedure was used with AVIDx-AP™(alkaline phosphatase; Tropix) and AMPPD substrate, according to themanufacturer's recommended protocol. Chemiluminescence was detectedusing a Camera Luminometer (Tropix) and Polaroid 612 film. Comparison ofresults from reactions with and without T7 SEQUENASE® showed that biotinhad been incorporated into DNA and was easily detectable by using achemiluminescence assay. If detection uses an indirect label detectionprocess (i.e., biotin label reacted with AVIDx-AP™ for detection, ratherthan direct detection of incorporated label, such as FITC) and thedetection step is done on beads DYNABEADS®, Dynal; oligo(dT) beads,Promega!, or on a matrix such as oligo(dT) cellulose, Stratagene POLY(A)QUICK™!, the capture matrix must be incubated with some agent to blockthe non-specific sticking of the detection reagent to the matrix,I-Block reagent mix (Blocking Buffer: SOUTHERN-LIGHT™, Tropix) has beenused for this purpose. Washed beads or oligo(dT) cellulose!, withattached DNA were washed 1× in Blocking Buffer then incubated inBlocking Buffer for 10 to 30 min, before washing and addition ofAVIDx-AP™ (Tropix). Excess unbound AVIDx-AP™ was then removed by washingaccording to the Tropix protocol. The capture matrix was then washedwith a buffer compatible with substrate detection (Assay Buffer, Tropixcan be used for detection by chemiluminescence; alternatively fordetection by fluorescence, the ATTOPHOS™ system of JBL Scientific, withits compatible buffers can be used). Substrate is added and theDNA-bound AP is detected by the appropriate means.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 500 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Lambda                                                          (viii) POSITION IN GENOME:                                                    (A) CHROMOSOME/SEGMENT: 500 base pairs                                        (B) MAP POSITION: 7131 to 7630                                                (C) UNITS: bp                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GATGAGTTCGTGTCCGTACAACTGGCGTAATCATGGCCCTTCGGGGCCATTGTTTCTCTG60                TGGAGGAGTCCATGACGAAAGATGAACTGATTGCCCGTCTCCGCTCGCTGGGTGAACAAC120               TGAACCGTGATGTCAGCCTGACGGGGACGAAAGAAGAACTGGCGCTCCGTGTGGCAGAGC180               TGAAAGAGGAGCTTGATGACACGGATGAAACTGCCGGTCAGGACACCCCTCTCAGCCGGG240               AAAATGTGCTGACCGGACATGAAAATGAGGTGGGATCAGCGCAGCCGGATACCGTGATTC300               TGGATACGTCTGAACTGGTCACGGTCGTGGCACTGGTGAAGCTGCATACTGATGCACTTC360               ACGCCACGCGGGATGAACCTGTGGCATTTGTGCTGCCGGGAACGGCGTTTCGTGTCTCTG420               CCGGTGTGGCAGCCGAAATGACAGAGCGCGGCCTGGCCAGAATGCAATAACGGGAGGCGC480               TGTGGCTGATTTCGATAACC500                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Cleavage site for Dpn I                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GATC4                                                                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: lambda                                                          (C) INDIVIDUAL ISOLATE: PCR primer from lambda genome                         (viii) POSITION IN GENOME:                                                    (B) MAP POSITION: 6631 to 6655                                                (C) UNITS: bp                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCGGCACGGAGTGGAGCAAGCGTGA25                                                   (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: lambda                                                          (C) INDIVIDUAL ISOLATE: polyA construct from lambda genome                    (viii) POSITION IN GENOME:                                                    (B) MAP POSITION: 8001-8023                                                   (C) UNITS: bp                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AAAAAAAAAAAAAAAATACGGCTGAGGTTTTCAACGGC38                                      __________________________________________________________________________

It is claimed:
 1. A diagnostic method for detecting a linear duplex DNAanalyte, having first and second strands, containing a first internalDNA target sequence, comprisingproviding a set of two DNA probes, havingfirst and second probe strands, where the first and second probe strands(i) contain complementary sequences to the first and second targetsequence strands, and (ii) where these complementary sequences alsocontain complementary overlap between the probe strands, coating theprobes with RecA protein in a RecA protein coating reaction, saidcoating reaction containing a nucleotide cofactor selected from thegroup consisting of ATPγS, rATP, dATP, and GTPγS, or a mixture ofnucleotide cofactors consisting of ATPγS and rATP or ATPγS and ADP,combining the RecA coated probes with the linear duplex DNA, whichcontains the target sequence, under conditions that produce aprobe:target complex containing the probe strands and both targetstrands, where said complex is stable to deproteinization, and detectingthe presence of the probe DNA in the probe:target complex.
 2. The methodof claim 1, wherein said DNA probes are prepared by nick-translation. 3.The method of claim 1, wherein said cofactor is rATP and said reactingis carried out in the presence or absence of an ATP regenerating system.4. The method of claim 1, where said cofactor is dATP.
 5. The method ofclaim 1, which further comprises providing a second set of two DNAprobes, said probes prepared by nick-translation and having first andsecond probe strands, complementary to a second duplex target sequence,where the first strand of the probe contains sequences complementary toone strand of the second target sequence and the second strand of theprobe contains sequences complementary to the other strand of the secondtarget sequence, where (i) these probes also have a region ofcomplementary overlap to each other, and (ii) the second set of probesdoes not hybridize to the first set of probes.
 6. The method of claim 1,where said coating reaction contains ATP.
 7. The method of claim 6,wherein said DNA probes are prepared by nick-translation.
 8. A methodfor isolating a linear duplex DNA analyte, having first and secondstrands, containing a first internal DNA target sequence, where saidduplex DNA analyte is present in a mixture of nucleic acid molecules,comprisingproviding a set of two DNA probes, having first and secondprobe strands, where the first and second probe strands (i) containcomplementary sequences to the first and second target sequence strands,and (ii) where these complementary sequences also contain complementaryoverlap between the probe strands, coating the probes with RecA proteinin a RecA protein coating reaction, combining the RecA coated probeswith the linear duplex DNA, which contains the target sequence, underconditions that produce a probe:target complex containing the probestrands and both target strands, where said complex is stable todeproteinization, separating the probe:target complex from the mixtureof nucleic acid molecules, and isolating the duplex DNA analytecontaining the target sequence from the probe:target complex.
 9. Themethod of claim 8, wherein said DNA probes are prepared bynick-translation.
 10. The method of claim 8, wherein the probes arebound to a solid support.
 11. The method of claim 8, wherein said probescontain at least one biotin moiety.
 12. The method of claim 11, wheresaid separating is accomplished using streptavidin.
 13. The method ofclaim 12, where the streptavidin is bound to a solid support.
 14. Themethod of claim 8, wherein said isolating further includes heatdenaturation of the probe:target complex at a temperature (i) sufficientto release the duplex DNA analyte containing the target sequence fromthe complex, and (ii) below the melting temperature of the duplex DNAanalyte containing the target sequence.
 15. The method of claim 14,wherein the duplex DNA analyte is denatured into single-stranded DNA.16. The method of claim 8, wherein said isolating further includes heatdenaturation of the probe:target complex at a temperature (i) sufficientto release the duplex DNA analyte containing the target sequence fromthe complex, and (ii) at or above the melting temperature of the duplexDNA analyte containing the target sequence.
 17. A method for detecting alinear duplex DNA analyte, having first and second strands, containing afirst internal DNA target sequence, where said duplex DNA analyte ispresent in a mixture of nucleic acid molecules, comprisingisolating thelinear duplex DNA analyte as described in claim 8, wherein saidisolating further includes heat denaturation of the probe:target complexat a temperature (i) sufficient to release the duplex DNA analytecontaining the target sequence from the complex, and (ii) at or abovethe melting temperature of the duplex DNA analyte containing the targetsequence, adding at least one DNA synthesis primer, which iscomplementary to the target sequence and has 5' and 3' ends, where saidprimer does not contain sequences that were present in either of the twoDNA probes, and where the detection of the DNA analyte is accomplishedby DNA polymerase facilitated primer extension from the 3'-end of theprimer, wherein the primer extension is performed in the presence of allfour dNTPs and at least one dNTP contains a detectable moiety.
 18. Themethod of claim 17, wherein said DNA probes are prepared bynick-translation.
 19. The method of claim 17, wherein said primer strandcontains an end terminal extension of DNA that is not complementary toeither target strand.
 20. The method of claim 17, wherein at least oneDNA synthesis primer contains a capture moiety.
 21. The method of claim20, where said detection further includes the generation of primerextension products containing the capture moiety and the detectionmoiety and said products are isolated using said capture moiety.